Selective inhibition of internally initiated RNA translation

ABSTRACT

PCT No. PCT/US95/12615 Sec. 371 Date Oct. 6, 1997 Sec. 102(e) Date Oct. 6, 1997 PCT Filed Oct. 11, 1995 PCT Pub. No. WO96/11211 PCT Pub. Date Apr. 18, 1996A method to inhibit translation of an mRNA, which is intitiated at an internal ribosome entry site of the mRNA and requires binding of a protein factor to that site, is disclosed. The method comprises a step of providing, in an in vitro, or in vivo system that is capable of translating the mNRA, an inhibitory effective amount of a molecule that selectively binds to the protein factor, thereby preventing that factor from binding to the mNRA. The inhibitor molecule is an RNA oligonucleotide consisting of less than 35 nucleotides or a structural mimic of such an RNA oligonucleotide. Nucleotide sequences of such inhibitor RNA oligonucleotides include portions of the following sequences: the 60 nucleotide sequence of a yeast inhibitor RNA or of the sequence complementary to that yeast inhibitor RNA; nucleotides 186-220 of poliovirus (stem-loop D); nucleotides 578-618 of poliovirus (stem-loop G); nucleotides 260-415 of poliovirus (stem-loop E); nucleotides 448-556 of poliovirus (stemp-loop F); and the sequence of the internal ribosome entry site of the immunoglobulin heavy chain binding protein (Bip).

This application is a 371 of PCT/US95/12615, and a continuation-in-partof application U.S. Ser. No. 08/321,427 filed 11 Oct. 1994, thedisclosure of which is incorporated by reference in its entirety. Thisinvention was made with funding from National Institutes of Health GrantNos. AI-18272, AI-27451 and AI-38056. The U.S. Government has certainrights in this invention.

TECHNICAL FIELD

The invention relates to selective inhibition of translation of certainmRNAs. More particularly, the invention relates to selective inhibitionof an mRNA which is initiated at an internal ribosome entry site, suchas a picornavirus RNA, by a small RNA or a molecular mimic thereof. ThisRNA or mimic interacts specifically with a cellular protein to preventbinding of that protein to the internal ribosome entry site, therebyinhibiting translation initiation at that entry site.

BACKGROUND ART

Picornaviruses include inter alia polioviruses, which cause infantileparalysis, and rhinoviruses, which cause the common cold.Picorna-related viruses, which replicate by mechanisms similar topicornaviruses, include hepatitis A and C, major causes of humanhepatitis. Although poliovirus vaccines are available, cases of poliostill develop where vaccination is not properly used. Vaccines for otherpicornaviruses may not be feasible, for instance, due to the high rateof mutability of the viral coat proteins in the rhinoviruses. Therefore,there is a need for methods and compositions for selectively inhibitingpicornavirus replication without toxic effects on the host cells.

Poliovirus, the prototype member of the picornaviridae family, is asingle stranded, plus-sense RNA virus which multiplies in the cytoplasmof infected cells. The RNA genome comprises approximately 7,500nucleotides and codes for a 250 kDa polyprotein (Kitamura, N. et al.Nature (1981) 291:547-553 and Racaniello, V. R., et al. Proc Natl AcadSci USA (1981) 78:4887-4891. The unusually long 5' untranslated region(5'UTR) of poliovirus RNA (750 nucleotides) is highly structured(Skinner, M. A. et al. J Mol Biol (1989) 207:379-392; Agol, V. Adv VirusRes (1991) 40:103-180) and contains six to eight upstream AUGs, none ofwhich appears to be used in initiation of translation (Pelletier, J. etal. J Virol (1988a) 62:4486-4492.

Translation of most mammalian cellular mRNAs proceeds by binding ofribosomes to the 5' cap structure followed by scanning of the mRNA untilthe appropriate AUG is encountered by the ribosome (Kozak, M. MicrobiolRev (1983) 47:1-45). In contrast translation of naturally uncappedpoliovirus RNA has been shown to be mediated by a mechanism involvinginternal entry of ribosomes near the initiator AUG (Pelletier, J. et al.Nature (1988) 334:320-325). Recent studies have demonstrated thatinternal entry of ribosomes requires an element located betweennucleotides 320-631 within the 5'UTR of poliovirus RNA (Pelletier, J. etal., supra). This sequence element has been termed a ribosome landingpad (RLP) or, more generally, internal ribosome entry site (IRES).Although a number of cellular polypeptides have been implicated inIRES-dependent translation, the precise mechanism of internal initiationof translation remains poorly understood.

In addition to poliovirus many other picornaviruses have been shown toutilize this novel mechanism for initiation of translation (Jang, S. K.et al. Genes Dev (1990) 4:1560-1572, Belsham, G. J. et al. J Virol(1990) 64:5389-5395, Jackson, R. et al. Trends Biochem Sci (1990)15:477-483, Luz, N. et al. FEBS Letters (1990) 269:311-314, Luz, N. etal. Virology (1991) 65:6486-6494, Bandopadhyay, P. K. et al. J Virol(1992) 66:6249-6256, Borman, A. et al. Virology (1992) 188:685-696,Borman, A. et al. Gen Virol (1993) 74:1775-1788). The RNA genomes of twopicorna-related viruses, hepatitis A and C, have been shown to utilizeinternal ribosome entry for translation initiation (Kohara, K. T. et al.J Virol (1992) 66:1476-1483 and Glass, M. J. et al. Virology (1993)193:842-852). Two cellular mRNAs, encoding immunoglobulin heavy chainbinding protein (Bip), the mouse androgen receptor (32) and theantennapedia of Drosophila, also have been shown to use internalinitiation of translation (Macejak, D. G. et al. Nature (1991) 353:90-94and Oh, S. K. et al. Genes Dev (1992) 6:1643-1653).

All picornaviral mRNAs that utilize IRES-dependent translation contain apolypyrimidine tract located at the 3'-border of the IRES sequenceswithin the 5'UTR. Recent evidence indicates that proper spacing betweenthe polypyrimidine tract and the cryptic AUG at nucleotide 586 of thepoliovirus 5'UTR is important for viral translation (Jackson et al.(1990, supra), Jang et al. (1990, supra), Pilipenko, E. V. et al. Cell(1992) 68:119-131).

Accurate translation of poliovirus mRNA in rabbit reticulocyte lysaterequires HeLa cell proteins, indicating involvement of cellular proteinsin internal initiation of translation (Brown, B. A. et al. Virology(1979) 97:376-405; Dorner, H. A. et al. J Virol (1984) 50:507-514) . A50 kDa protein has been shown to interact with the RNA stem-loopstructure located between nucleotides 186-221 in poliovirus type 1 RNA(Najita, L. Proc Natl Acad Sci USA (1990) 87:5846-5850). Thephysiological significance of this binding is yet unclear.

Another protein called p52, more abundant in HeLa cells than in rabbitreticulocytes, has been found to specifically bind to the stem-loopstructure between nucleotides 559-624 of type 2 poliovirus RNA(Meerovitch, K. et al. Genes Dev (1989) 3:1026-1034). This p52 proteinappears to be identical to the human La auto antigen (Meerovitch, K. etal. J Virol (1993) 67:3798-3807). This nuclear protein, which isrecognized by antibodies from patients with the autoimmune disorderlupus erythematosus, leaches out of the nucleus into the cytoplasm inpoliovirus-infected HeLa cells. Cell extracts immunodepleted with Laantibodies fail to promote cap-independent translation and exogenousaddition of purified La protein corrects aberrant translation ofpoliovirus RNA in reticulocyte lysate which contains little or no p52(Meerovitch et al. (1993, supra).

UV crosslinking studies have demonstrated another cellular protein, p57to interact with IRES elements of encephalomyocarditis (EMC),foot-and-mouth disease, rhino-, polio- and hepatitis A viruses (Jang etal. 1990, supra; Borovjagin, A. V. et al. Nucleic Acids Res (1991)19:4999-5005; Luz et al. 1991, supra; Pestova, T. V. et al. J Virol(1991) 65:6194-6204; Borman et al. 1993, supra, and Chang, K. H. et al.J Virol (1993) 67:6716-6725). It has been demonstrated recently that p57binding to an IRES of EMCV is identical to that of a polypyrimidinetract binding protein (PTB), which presumably plays a role in a nuclearsplicing (Hellen, C. U. T. et al. Proc natl Acad Sci USA (1993)90:7642-7646). Anti-PTB antibody inhibits translation of EMCV andpoliovirus RNA and, therefore, PTB may be directly involved inIRES-directed translation.

In addition two other cellular proteins with molecular weights of 38 and48 kDa have been shown to specifically interact with RNA structuresspanning nucleotides 286-456 of poliovirus. These two proteins arereported to be present in HeLa cells in higher quantities than inreticulocyte lysate and appear to be involved specifically in poliovirustranslation (Gebhard, J. R. et al. J Virol (1992) 66:3101-3109). Another54 kDa protein cross-links to a region between nucleotides 456-626 andis required for translation of all mRNAs (Gebhard et al. 1992, supra). Arecent report indicates the role of a 97 kDa protein in IRES-dependenttranslation of human rhinovirus RNA (Borman et al. 1993, supra).RNA-protein complex formation has also been demonstrated with theregions encompassing nucleotides 98-182 and 510-629 of the poliovirusRNA (del Angel, P. A. G. et al. Proc Natl Acad Sci USA (1989)86:8299-8303).

Taken together, the results above are compatible with a mechanism ofpicornaviral translation that involves direct interaction betweencellular factors and RNA sequences and/or secondary structures leadingto internal initiation. The action of the binding proteins in thismechanism is not known, but transacting proteins may direct ribosomes toenter the mRNA or may alter RNA structure to facilitate ribosomebinding.

In a previous study the present inventors have shown that yeast cellsare incapable of translating poliovirus RNA both in vivo and in vitroand that this lack of translation represents selective translationinhibition which requires the 5'UTR of the viral RNA (Coward, P. et al.J Virol (1992) 66:286-295). The inhibitory effect was found to be due toa transacting factor present in yeast lysate that can also inhibit theability of HeLa cell extracts to translate poliovirus RNA. Initialcharacterization of this inhibitor showed that its activity was heatstable, resistant to proteinase K digestion, phenol extraction and DNasedigestion, but sensitive to RNase (Coward et al. 1992, supra).

DISCLOSURE OF THE INVENTION

The present invention is directed to methods and compositions forinhibiting translation of an mRNA, such as poliovirus RNA, which isinitiated at an internal ribosome entry site and requires binding of aprotein factor to that site. The invention is based on theidentification of an RNA of 60 nucleotides from the yeast S. cerevisiaewhich inhibits internally initiated translation but not cap-dependenttranslation. The yeast inhibitor RNA (I-RNA) binds to various cellularproteins that are reported to be involved in internal initiation oftranslation, competing with the 5'UTR of poliovirus RNA for binding tosuch proteins, and selectively inhibiting translation of viral mNRAwithout affecting host cell protein synthesis. When expressed in hostcells, the inhibitor RNA specifically and efficiently inhibitstranslation of poliovirus RNA and thereby protects these cells fromviral infection. Analyses of structural requirements of this RNA forinhibition of translation has enabled the design of substantiallysmaller RNA inhibitors of internally-initiated RNA translation and,ultimately, design of non-RNA molecular mimics of such inhibitor RNAs.

Thus, in one aspect, the invention is directed to a method to inhibittranslation of an mRNA, which translation is initiated at an internalribosome entry site of the mRNA and requires binding of a protein factorto that site. This method comprises a step of providing, in a systemthat is capable of translating this mRNA, an inhibitory effective amountof a molecule that selectively binds to the required protein factor,thereby preventing that factor from binding to the internal ribosomeentry site of the mRNA. In preferred embodiments the inhibitor moleculeis an RNA oligonucleotide consisting of less than 35 nucleotides or astructural mimic of such an RNA oligonucleotide.

In other aspects the invention is directed to an expression constructencoding an RNA molecule comprising an RNA oligonucleotide consisting ofless than 35 nucleotides linked to a heterologous nucleotide sequence,and to an inhibitor molecule suitable for use in the method oftranslation inhibition of the invention, which provides thethree-dimensional array of intermolecular forces exhibited by aninternal ribosome entry site of an mRNA.

More particularly, the invention relates to a method to inhibittranslation of an mRNA, which translation is initiated at an internalribosome entry site of the mRNA and requires binding of a protein factorto said site, which method comprises: a step of providing, in a systemthat is capable of translating the subject mNRA, a translationinhibitory effective amount of a molecule that selectively binds to thefactor, thereby preventing the factor from binding to the site of themRNA, wherein the molecule is selected from the group consisting of: anRNA oligonucleotide consisting of less than 35 nucleotides; and astructural mimic of said RNA oligonucleotide.

In a preferred embodiment of the method, the mNRA is a viral RNA of avirus selected from the group consisting of picornaviruses,flaviviruses, coronaviruses, hepatitis B viruses, rhabdoviruses,adenoviruses, and parainfluenza viruses. In particular, the virus may beselected from the group consisting of polioviruses, rhinoviruses,hepatitis A viruses, coxsackie viruses, encephalomyocarditis viruses,foot-and-mouth disease viruses, echo viruses, hepatitis C viruses,infectious bronchitis viruses, duck hepatitis B viruses, human hepatitisB viruses, vesicular stomatitis viruses, and sendai viruses.Alternatively, the mRNA to be inhibited may be a cellular mNRA with aninternal ribosome entry sites, such as a cellular mRNA encoding animmunoglobulin heavy chain binding protein (Bip).

In the method of the invention, the inhibitor molecule may be providedby adding the RNA oligonucleotide of the invention to the system that iscapable of translating the mRNA. Alternatively, the molecule is providedby adding to the system that is capable of translating the mRNA an RNAmolecule comprising the RNA oligonucleotide linked to a heterologousnucleotide sequence. The RNA oligonucleotide also may be provided by anexpression construct for in situ production of the RNA oligonucleotidein the system that is capable of translating the subject mRNA.

The system to be inhibited by the invention method, that is capable oftranslating the subject mRNA, may be a cell-free system or a host cellthat is infected or at risk of infection with a virus which produces thesubject mRNA. The host cell may be a mammalian cell, either in a cellculture or in a host animal in which translation of the subject mRNA isto be inhibited.

In another aspect the invention relates to a molecule that inhibitstranslation of an mRNA, which translation is initiated at an internalribosome entry site of this mRNA and requires binding of a proteinfactor to that site. This molecule selectively binds to the factor,thereby preventing the factor from binding to the ribosome entry site ofthe mRNA. The invention molecule is selected from the group consistingof an RNA oligonucleotide consisting of less than 35 nucleotides; and astructural mimic of such an RNA oligonucleotide. In preferredembodiments, this molecule is an RNA oligonucleotide having a sequencewhich comprises at least one portion selected from the group ofsequences consisting of the sequence shown in FIG. 1 A; a sequencecomplementary to the sequence shown in FIG. 1A; the sequence ofnucleotides 186-220 of poliovirus (stem-loop D); the sequence ofnucleotides 578-618 of poliovirus (stem-loop G); the sequence ofnucleotides 260-415 of poliovirus (stem-loop E); the sequence ofnucleotides 448-556 of poliovirus (stem-loop F); and the sequence of animmunoglobulin heavy chain binding protein (Bip) mRNA which binds saidprotein factor to said internal ribosome entry site of said mNRA. In amore preferred embodiment, the nucleotide sequence of the RNAoligonucleotide is the ribonucleotide sequence 5'GCGCGGGCAGCGCA 3'(SEQID:1). In other aspects the invention is related to an RNA moleculecomprising an RNA oligonucleotide of claim 10 linked to a heterologousnucleotide sequence and an expression construct encoding an RNA moleculewherein the RNA molecule comprises an RNA oligonucleotide of theinvention linked to a heterologous nucleotide sequence.

The invention also provides screening assays for identifying moleculesthat inhibit translation of an mRNA, which translation is initiated atan internal ribosome entry site of this mRNA and requires binding of aprotein factor to that site. This inhibitor molecule selectively bindsto the translation initiation factor, thereby preventing the factor frombinding to the ribosome entry site of the mRNA. Assays to identifyinitiation factor binding molecules of the invention include immobilizedligand binding assays, solution binding assays, scintillation proximityassays, di-hybrid screening assays, and the like.

In preferred embodiments of the method and molecules of the invention,the protein factor is a 52 kDa La autoantigen. In addition, three otherhuman cellular polypeptides of apparent molecular masses of 80, 70 and37 kDa may be used to detect molecules which exhibit the translationalinhibitory activity of I-RNA of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1D shows the nucleotide sequence, cloning, expression andactivity of the exemplary yeast translation inhibitor RNA (I-RNA). FIG.1A: Sequence of the 60-nucleotide purified yeast inhibitor RNA [SEQ IDNO:2] was determined as described in materials and methods. 5'- and3'-termini of the RNA are indicated. FIG. 1B: schematic Illustration ofthe pSDIR plasmid expressing I-RNA. The position of HindIII and EcoRIrestriction endonuclease sites are shown. T7 and SP6 indicate thelocation of the respective promoters and the arrows show the directionof transcription. FIG. 1C: The I-RNA (sense transcript) was transcribedin vitro using T7 RNA polymerase from plasmid pSDIR linearized withHindIII restriction enzyme. Four micrograms of the synthesized RNA wasthen mixed with denaturing gel loading dye (US Biochemicals), heated at55° C. for 10 min and then analyzed on a 1.2% agarose gel along with the1 kb ladder DNA (BRL) marker (lane M) under conditions for analysis ofDNA samples. The position of the I-RNA band on the gel is indicated.FIG. 1D: In vitro translations of pG3CAT and p2CAT RNAs in HeLacell-free translation lysates were performed in the absence or presenceof the inhibitor RNA. Two μg of pG3CAT or P2 CAT RNAs were added perreaction containing 80 μg of HeLa cell lysates. Approximately 4 μg ofpartially purified yeast inhibitor RNA and 1 μg of synthetic inhibitorRNA were used in respective reactions indicated in the figure. Thelocation of the CAT gene product is shown with an arrowhead.

FIGS. 2A-2D illustrate the requirement of the poliovirus 5'UTR sequencefor inhibition of translation by the yeast inhibitor RNA. FIGS. 2A, 2Band 2C: HeLa cell-free translation lysates were used to translate theRNAs listed above the lanes in each panel. In vitro translations wereperformed with approximately 2 μg of either capped or uncapped RNA asindicated for each deletion mutant construct, in the absence or presenceof 1 μg purified I-RNA. Each reaction contained 80 μg of HeLa celllysate protein. The position of the CAT protein is indicated at the leftof FIG. 2A. FIG. 2D: The diagram shows poliovirus 5'-UTR deletion mutantconstructs that were used for the above experiment. Vertically hatchedboxes represent SP6 RNA polymerase promoters. Solid black boxesrepresent the sequences from the poliovirus 5'-UTR, and diagonallyhatched boxes indicate CAT gene coding sequences. The number underneaththe plasmids represents the nucleotide at the edge of the deletion.

FIG. 3 illustrates formation of a complex between the I-RNA and cellularproteins which retards the I-RNA during gel electrophoresis and iscompetitively inhibited by the 5'-UTR of the poliovirus RNA. ³²P-labeled I-RNA probe was incubated with or without HeLa S-10 extract inthe binding reactions as described in the examples below. TheRNA-protein complexes were analyzed on a nondenaturing 4% polyacrylamidegel. Panel A shows the mobility shift from free probe (FP) in absence ofthe S-10 extract (lane 2) to the complexed form (C) in presence of theS-10 extract. Panel B shows the results of the competition experimentswith unlabeled competitor RNAs. Ten, 50 and 100-fold molar excesses ofunlabeled I-RNA (lanes 3-5) or unlabeled 5'-UTR (lanes 7-9) were used inthe binding reactions. The reaction in lane 10 contained loo-fold molarexcess of unlabeled nonspecific RNA (NSP).

FIGS. 4A-4B show that the I-RNA binds proteins that interact withpoliovirus 5'-UTR. UV crosslinking of the ³² p-labeled I-RNA with HeLacell proteins was performed as described in the examples below. FIG. 4A:The numbers at left refer to the approximate molecular masses of theproteins that interact with I-RNA. For competition studies a 100-foldmolar excess of the respective unlabeled competitor RNA was added ineach binding reaction. The competitor RNAs used were: I-RNA (lane 3),5'-UTR (lane 4) and nonspecific (Nsp) RNA (lane 5). FIG. 4B: The numbersat left refer to the molecular mass of the protein markers (BRL) (laneM). The numbers at right refer to the molecular mass of each proteinwhich crosslinks to labeled I-RNA probe. 100-fold molar excess of theunlabeled competitor RNAs such as I-RNA (lane 3), UTR 559-624 RNA (lane4), and nonspecific (Nsp) RNA (lane 5) were used in the bindingreactions.

FIG. 5 illustrates the predicted secondary structure of the 5'UTR ofpoliovirus RNA showing possible interaction sites with cellular proteinsby different structural domains. The molecular masses of the cellularproteins with their possible sites of interactions (indicated by thenucleotides within parentheses) are shown. The figure is a modifiedversion of secondary structure predictions published by Pilipenko et al.(1992, supra), Jackson et al. (1990, supra) and Dildine, S. L. et al. JVirology (1992) 66:4364-4376.

FIGS. 6A-6B illustrate UV crosslinking of HeLa cell proteins to ³²P-labeled I-RNA, 5'UTR RNA, stem-loop SL-G and SL-D RNA probes. FIG. 6A:The numbers at left refer to the molecular mass of the protein markers(lane M). Individual ³² p-labeled RNA probes such as I-RNA (lanes 1, 2),5'UTR RNA (lanes 3, 4), stem-loop G RNA (lanes 5, 6) and stem-loop D RNA(lanes 7, 8) were incubated either with (lanes 2, 4, 6, 8) or without(lanes 1, 3, 5, 7) HeLa S-10 extract in the binding reactions followedby UV crosslinking and gel analysis. The numbers at right of FIG. 6Adenote the approximate molecular masses of the crosslinked proteins.FIG. 6B: ³² P-labeled stem-loop SL-G (UTR 559-624) and SL-D (UTR178-224) RNA in lanes 1 and 2, respectively, were incubated with HeLaS-10 extract, crosslinked and analyzed side by side to compare themobilities of the crosslinked proteins. The numbers at right refer tothe estimated molecular masses of the proteins indicated with thearrowheads.

FIGS. 7A-7C show that I-RNA competes with both stem-loops SL-G and SL-Dfor protein binding. The ³² P-labeled RNA probes used in theUV-crosslinking experiments are listed on top of each panel. The numbersat left of FIG. 7A indicate the molecular masses of protein markers inlane M. FIG. 7A: Lane 1, no extract; lane 2, extract with no unlabeledcompetitor RNA; lane 3, unlabeled 5'UTR competitor; lane 4, unlabeledI-RNA competitor; lane 5, unlabeled stem-loop SL-G (i.e., UTR 559-624);lane 6, unlabeled SL-D (i.e., UTR 178-224); lane 7, unlabelednonspecific RNA; lane 8, unlabeled SL-B RNA (i.e., UTR 51-78); lane 9,unlabeled SL-C RNA (i.e., UTR 124-162). FIG. 7B: Lane 1, no extract;lane 2, extract with no unlabeled RNA;

lane 3, unlabeled 5'SL-G RNA competitor; lane 4, unlabeled I-RNAcompetitor; lane 5, unlabeled 5'SL-D RNA competitor; lane 6, unlabelednonspecific RNA competitor; lane 7, unlabeled SL-B RNA; lane 8,unlabeled SL-C RNA. FIG. 7C: Lane 1, no extract; lane 2, extract with nounlabeled competitor; lane 3, unlabeled SL-D competitor; lane 4,unlabeled I-RNA competitor; lane 5, nonspecific RNA; lane 6, unlabeledSL-G competitor; lane 7, unlabeled SL-B RNA; lane 8, unlabeled SL-C RNA.The arrowheads indicate protein-nucleotidyl complexes with proteins ofmolecular masses of 52, 54 and 57 kDa respectively, from bottom to top.

FIGS. 8A-8B demonstrate that I-RNA inhibits internal initiation oftranslation in vitro. FIG 8A: The construct pBIP-LUC containing the5'UTR of Bip mNRA linked to a reporter gene (luciferase) was translatedin vitro in HeLa cell lysates in the absence (lane 1) or presence (lane2) of the yeast inhibitor. As a control the construct pG3CAT was alsotranslated in the absence (lane 3) or presence (lane 4) of the yeastinhibitor. The products were analyzed on a SDS-14% polyacrylamide gel.The arrowhead at the left indicates the position of the luciferase geneproduct (LUC) and the arrowhead to the right indicates the product ofCAT gene (CAT). FIG. 8B: A bicistronic construct pPB310 containing theCAT gene and luciferase gene flanked by TMEV 5'UTR was translated invitro in HeLa cell lysates in absence (lane 1) or presence (lane 2) ofI-RNA. The product were analyzed on a SDS-14% polyacrylamide gel. Thearrowheads at left denote the positions of the CAT gene product (CAT)and luciferase gene product (LUC).

FIG. 9 shows that I-RNA inhibits translation of poliovirus RNA in vivo.Monolayers of HeLa cells were transfected with viral RNA alone, I-RNAalone or viral RNA and I-RNA together. After transfection the cells werelabeled with ³⁵ S-methionine and in vivo labeled proteins were analyzedon a SDS-14% polyacrylamide gel either directly (panel A) or afterimmunoprecipitation with anticapsid antibody (panel B) as described inthe examples below. Panel A: The RNAs added to each transfectionreaction are as indicated in the figure. Panel B: The panel shows theimmunoprecipitated in vivo labeled proteins from the transfectionreactions shown in panel A, lanes 1-5. The positions of the polioviruscapsid proteins are indicated to the left of panel B.

FIGS. 10A-10B illustrate computer-predicted secondary structures of theyeast inhibitor RNA (SEQ ID NO:2). FIGS. 10A and 10B show two probablesecondary structures of the RNA. The numbers refer to the positions ofthe nucleotides from the 5'-end of the RNA. The free energy calculatedfor each predicted structure is given below the respective structure.

FIGS. 11A-11B show that yeast I-RNA specifically inhibits internalribosome entry site (IRES)-mediated translation in vitro. FIG. 11A:Inhibition by I-RNA of IRES-mediated translation from bicistronicconstructs in HeLa cell extracts. Synthesis of luciferase (Luc) isinitiated internally from virus IRES-elements and that of CAT isinitiated in a Cap-dependent manner (5'Cap-CAT-IRES-LUC 3'). Lanes 1, 4,5 and 7 did not contain I-RNA. Lanes 2, 3, 6 and 8 contained 1 μg ofI-RNA. FIG. 11B: Effect of I-RNA on in vitro translation mediated byvarious monocistronic RNAs of immunoglobulin heavy chain binding protein(Bip, lanes 1, 2), CAT (lanes 3, 4, 9, 10), P2 CAT (containing PV 5'UTR,lanes 7, 8), pGemLUC (lanes 5, 6), pCITE (containing EMCV IRES, lanes11, 12), and yeast α36 mRNA (lanes 13, 14). Lanes 1, 3, 5, 7, 9, 11, 13contained no inhibitor. Lanes 2, 4, 6, 8, 10, 12, 14 contained 1 μgI-RNA.

FIGS. 12A-12B show that the HeLa 52 kDa I-RNA-binding protein isidentical to La autoantigen. Two assays, gel retardation followed bysupershifting with La-antibody FIG. 12A, and UC-crosslinking followed byimmunoprecipitation with La-antibody FIG. 12B, were performed toidentify the 52 Kda I-RNA binding HeLa cell protein as the La antigen.

FIG. 13 shows that antisense I-RNA also binds a HeLa 52 kDa protein thatinteracts with UTR (559-624). UV crosslinking of 32p labeled I-RNA (lane1 and 2) or antisense I-RNA (lane 3 and 4) with HeLa cell free extract(S10) A 52 kDa protein is complexed to both I-RNA and anti I-RNA(indicated with an arrowhead) which can be competed by cold UTR 559-624(lane 2). Lane M contained 14C labeled protein molecular mass markerswith sizes of 43 and 29 kDa respectively from the top. (A) Gelretardation: ³² P-labeled I-RNA probe was incubated with 50 μg of HeLaS10 extract (lanes 2 and 3 ) or with 0.3 μg of purified La proteinexpressed from the clone (lanes 4 and 5) in the presence (lanes 3 and 5)or absence (lanes 2 and 4) of antibody of La protein. I-RNA-proteincomplexes formed (denoted by C) with HeLa S10 or purified La proteinwere both super shifted (indicated by SS) with anti-La antibody.Preimmune IgG did not alter the migration of the complex (C). (B) ³²P-I-RNA (lanes 1, 2 and 3) or 32p UTR 559-624 RNA (lanes 4, 5 and 6)were UV-crosslinked to HeLa S10 proteins (lanes 2 and 5) or purified Laprotein (lanes 3 and 6). After RNase digestion protein-nucleotidylcomplexes were immunoprecipitated using anti-La antibody. Preimmune IgGdid not recognize the 52 kDa (La)-I-RNA complex, but recognized anonspecific protein migrating at ˜110 kDa indicated by a question mark(data not shown).

FIG. 14 shows inhibitory activity of a 14 nucleotide long RNA (BI-RNA)containing I-RNA sequences in specifically inhibiting translation byinternal initiation (P2 CAT) but not cap dependent translation (CAT).The figure shows translation of CAT gene (indicated with an arrowhead)from P2 CAT (lanes 1-4) or pCAT (lane 5 and 6) construct in the absence(lane 1 and 5) or presence of either I-RNA (lane 2) or 1 μg (lane 3 and6) and 2 μg (lane 4) of BI-RNA.

FIGS. 15A-15B illustrate the sequence of an active 14 nucleotide BI-RNAcontaining I-RNA sequences (SEQ ID NO:1) in the context of thecomputer-predicted secondary structures of the yeast inhibitor RNA asshown in FIG. 10 (SEQ ID NO:2). The solid line in FIG. 15B encompassesthe 14 nucleotides of the BI-RNA which were shown to inhibit translationusing internal initiation as described in FIG. 14. In the BI-RNA,nucleotide 13 of the native I-RNA structure is linked directly tonucleotide 30, by conventional phosphodiester linkage.

FIG. 16 diagrams I-RNA deletion mutant constructs tested for translationinhibiting activity. The nucleotide positions of the mutation sites areindicated for each mutant. The names of the respective mutants arelisted at the far left.

MODES OF CARRYING OUT THE INVENTION General Description and Definitions

The practice of the present invention will employ, unless otherwiseindicated, conventional biochemistry, immunology, molecular biology andrecombinant DNA techniques within the skill of the art. Such techniquesare explained fully in the literature. See, e.g., Maniatis et al.,Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A PracticalApproach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N.Gait, ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins,eds., 1985); Transcription and Translation (B. Hames & S. Higgins, eds.,1984); Animal Cell Culture (R.

Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning(1984).

The following terminology will be used in accordance with thedefinitions set out below in describing the present invention.

DNA "control sequences" refers collectively to promoter sequences,ribosome binding sites, polyadenylation signals, transcriptiontermination sequences, upstream regulatory domains, enhancers, and thelike, which collectively provide for the transcription and translationof a coding sequence in a host cell.

A coding sequence is "operably linked to" control sequences whenexpression of the coding sequences is effected when the expressionsystem is contained in an appropriate host cell.

A "host cell" is a cell which has been modified to contain, or iscapable of modification to contain, an exogenous DNA or RNA sequence.This includes, for instance, a cell infected by a virus or a celltransformed by a recombinant DNA molecule.

A "heterologous" region of a DNA or RNA construct is an identifiablesegment of DNA or RNA within or attached to another nucleic moleculethat is not found in association with the other molecule in nature.

Identification of a Translation Inhibitor Molecule

The present invention relates to a method to inhibit translation of anmRNA, which translation is initiated at an internal ribosome entry siteof that mRNA and requires binding of a protein factor to said site. Themethod comprises a step of providing, in a system that is capable oftranslating the mRNA, a translation inhibitory effective amount of amolecule that selectively binds to the factor, thereby preventing thatfactor from binding to the ribosome entry site of the mRNA. Thetranslation inhibitor molecule of this invention is selected from thegroup consisting of an RNA oligonucleotide consisting of less than 35nucleotides and a structural mimic of said RNA oligonucleotide.

Identification of a translation inhibitor molecule according to thepresent invention is exemplified in the first instance by the isolationand determination of the 60 nucleotide sequence of a naturally occurringinhibitor RNA from the yeast S. cerevisiae. This RNA selectivelyinhibits internally initiated translation but not cap-dependenttranslation, for instance, of picornavirus mRNAs. The isolation andsequencing of this small inhibitor RNA (I-RNA) is described inExample 1. Preparation of a synthetic DNA clone encoding the sequence ofan inhibitor RNA, and production of the RNA from the synthetic clone bytranscription with T7 RNA polymerase, are illustrated in Example 2.These methods may be adapted to the production of other RNAoligonucleotides of the invention, using routine approaches which arewell known in the art.

Selective inhibition of translation initiated by internal ribosome entrywithout substantially affecting cap-dependent translation, according tothe invention, may be demonstrated conveniently by in vitro methodsusing recombinant RNA constructs comprising both IRES- and5'cap-mediated translation initiation sites. See, for instance, Example3. Alternatively or in addition, selective inhibition of internallyinitiated translation may be demonstrated in vitro using recombinantbicistronic mRNA constructs of another viral mRNA, such as thosedescribed in Example 7, or of an internally initiated cellular mRNA,such as an mRNA encoding an immunoglobulin heavy chain binding proteinas illustrated in Example 8.

The translation inhibitor molecule of the invention selectively inhibitstranslation from an internal ribosome entry site by binding to a proteinfactor which is required for initiation of translation at the ribosomeentry site, thereby preventing that factor from binding to the ribosomeentry site of the mNRA. Such binding of the inhibitor molecule may beusing competitive binding methods to show disruption of complexesbetween the required protein factors and a selected mRNA, such as thosedescribed in Example 4 for disruption of complexes between poliovirusRNA sequences and HeLa host cell protein factor by the exemplary yeastI-RNA of the invention. In addition direct binding of an inhibitormolecule of the invention to protein factors required for internalinitiation of translation may be demonstrated conveniently using, forinstance, the UV-crosslinking method described for the yeast I-RNAmolecule in Example 5.

The ability of an inhibitor molecule of the invention to inhibittranslation of a viral mRNA in vivo may be demonstrated conveniently incell cultures as shown for inhibition of poliovirus RNA translation intransfected cells by the yeast I-RNA, which inhibits viral replicationand pathogenic effects, as illustrated in Example 9.

Accordingly, based on the general guidance and examples herein, one maydetermine using routine methods whether a given molecule exhibits theactivities of a translation inhibitor of the invention, namely,inhibition of translation of an mNRA, which translation is initiated atan internal ribosome entry site and requires binding of a protein factorto that site, by selectively binding to the factor, thereby preventingthe factor from binding to ribosome entry site of the subject mRNA.

Identification of Active RNA Oligonucleotides based on the Yeast I-RNA

In one preferred embodiment the translation inhibitor molecule of theinvention is an RNA oligonucleotide, based on the sequence of theexemplary yeast I-RNA, consisting of less than 60 nucleotides,preferably consisting of less than 35 nucleotides, more preferably lessthan 25 nucleotides, and still more preferably less than 15 nucleotides.As is known in the art it is advantageous to determine the minimumsequence of the I-RNA required for translation inhibition by means ofprotein factor binding because functional I-RNAs shorter than 60nucleotides offer greater efficiency in terms of production byconventional chemical synthesis and-in terms of their entry into intactcells by diffusion.

To determine which fragment or fragments of the yeast I-RNA exhibits thetranslation inhibitory activity according to the invention, conventionalgenetic engineering technology is used to prepare deletion mutants fromboth 5' and 3'-ends of I-RNA. Ten, 20 or 30 nucleotides are deleted at atime from either the 5' or 3' terminus of the I-RNA. RNA produced fromthese clones by transcription with T7 RNA polymerase is tested for theability to inhibit IRES-mediated translation but not cap-dependenttranslation, as described in the examples herein. Conventional methodsare also used to generate a nested set of deletions of 8-10 nucleotidesequences throughout the I-RNA molecule.

These systematic deletional approaches will identify sequences necessaryfor inhibition of viral translation and binding to host protein factors.Thus, such mutants which inhibit IRES-mediated translation will betested for loss of binding activity to protein factors such as the p52factor shown to bind to the yeast I-RNA or other factors mentionedhereinabove (for example p57) which are involved in I-RNA mediatedinhibition of viral translation.

As illustrated in FIG. 12, the p52 factor which binds to yeast I-RNA isidentical to the human La autoantigen as shown by immunological assays.This identity was further confirmed by both immunoprecipitationfollowing UV-crosslinking of the recombinant La protein to I-RNA and theability to supershift the La-I-RNA complex with anti-La antibody (FIG.12). That binding of La to I-RNA is relevant to translation inhibitionis indicated by the fact that purified recombinant La protein is able torestore PV IRES-mediated translation in the presence of the inhibitorRNA. Additional protein factors which bind to full-length or deletedI-RNAs and which can be used to identify other inhibitor molecules ofthe invention are described below.

A more selective mutational analysis also may be used to identify anactive oligonucleotide of the invention based on the larger sequence ofan active I-RNA such as the exemplary yeast I-RNA. In particular, it hasbeen found that an antisense RNA having the sequence exactlycomplementary to the sequence of yeast I-RNA is as efficient in bindingp52 as the sense I-RNA molecule. See FIG. 13. This result taken togetherwith the fact that there is no apparent sequence homology of the yeastI-RNA with poliovirus RNA sequences bind host cell protein factorsneeded for initiation of translation suggest that secondary structure ofI-RNA may play a crucial role in the inhibition of internal initiationof translation. Thus, many aspects of the secondary structure of asequence complementary to any RNA would be expected to be similar to thesecondary structure of the sequence itself, since generally the sameintrastrand base pairings would be able to form in the complementarystrand as in the original sequence.

Indeed, two computer-predicted secondary structures of the I-RNA havebeen generated that are thermodynamically relatively stable having ΔG of-27 and -21 Kcal/mol (FIG. 10). (These structures were predicted usingcommercially available software called DNASyS, but other similarsoftware is widely known and available. See, for instance Pilipenko etal. (1992, supra), Jackson et al. (1990, supra) and Dildine, S. L. etal. (1992, supra). These secondary structures partly resemble a p52binding site on the poliovirus mRNA.

Further, the secondary structure of the 60 nucleotide long native I-RNA(FIG. 10) does not change significantly by addition of 11 extranucleotides generated during the exemplary cloning procedure.Accordingly, by appropriate analysis of secondary structures, one canpredict whether linking of an active RNA oligonucleotide of theinvention to a heterologous sequence is likely to destabilize thesecondary structure of the oligonucleotide and thereby destroy itstranslation inhibitory activity. In addition the retention of therequired activity may be readily determined for any desired RNAoligonucleotide using the routine methods described herein.

By testing RNA oligonucleotides corresponding to various loops in thepredicted secondary structures of the yeast I-RNA, a 14 nucleotide longfragment of I-RNA was found to specifically inhibit poliovirusIRES-mediated translation. See FIG. 14. It should be noted that thetesting of RNA oligonucleotides comprising loops in computer-predictedsecondary enables identification of active RNA oligonucleotidescontaining noncontiguous portions of the larger I-RNA sequence, such asthe exemplary 14 nucleotide fragment which consists of nucleotides 7-13covalently coupled (by conventional 51-31 phosphodiester linkage) tonucleotides 30-36 of the yeast I-RNA.

Experimental results of a systematic deletional analysis of the yeastI-RNA are illustrated in Example 10, below. This analysis shows that theminimum I-RNA sequence required to inhibit PV IRES-mediated translationappears to reside between nucleotides 30-45. This conclusion issupported by two observations. First, a deletion mutant (I-3 RNA) whichcontains the entire I-RNA sequence except nucleotides 31-45 is totallyinactive in inhibiting viral IRES-mediated translation. Second, atruncated I-RNA (nt 30-45, I-9 RNA) retains considerable amount oftranslation-inhibitory activity.

However, a 25 nt long truncated RNA (I-7 RNA) containing the I-9 RNAsequence appears to be more active particularly in vivo. The shorter I-9RNA was only 50% as active as I-RNA in vivo. Both I-7 and I-9 RNAs canassume secondary structures having stem-and-loop sequences. Clearly,because of smaller size, I-9 RNA is much less stable than I-RNA whichmay affect stability of I-9 RNA inside a cell. Known thio-derivatives orother nuclease-resistant nucleotide analogs may be used to increasestability and thus activity of I-9 RNA or other inhibitor RNAs of theinvention which are exogenously provided to cells. The structure(s) ofI-RNA or its truncated derivatives may be important in IRES-mediatedtranslation inhibition. The fact that addition of an extra tennucleotides to the 3'-end of I-7 RNA (nt 26-50) significantly reducesits (1-6 RNA, nt. 26-60) translation-inhibitory activity may beindicative of alteration of structure of this RNA which should beavoided in designing the 3'-end of an inhibitor RNA of the invention.Similarly addition of another 5 nucleotides to the 5'-end of I-6 RNAdrastically reduces its (I-5, nt 20-60) ability to inhibit translation,indicating a need to consider such 5'-end effects in inhibitor RNAdesign.

Another alternate approach to identify a sequence and secondarystructure responsible for translation inhibition, for instance by p52binding, is to determine if a domain of I-RNA bound to p52 is resistantto RNase digestion according to routine methods known in the art. Inthis approach ³² P-body labeled I-RNA is incubated with purified pR2under binding conditions. The resulting complex is digested withmicrococcal nuclease or a mixture of RNases T1, T2, and A. The mixtureis then analyzed for one or more protected fragments following phenolextraction and ethanol precipitation. Protected fragment of 1-RNA aresequenced directly, for instance, using a commercially availablesequencing kit. An alternate sequencing approach is to hybridize theprotected fragment with cDNA encoding the I-RNA, followed by digestionof single stranded regions of the hybrid and sequence determination ofthe protected DNA fragment which is comparatively easier than RNAsequencing. The protected fragment is then tested for specificcompetition with unlabeled I-RNA but not with a non-specific RNA fortranslation inhibition and binding to the protein factor, as describedherein.

Besides the p52 La autoantigen protein, other protein factors have beenidentified which bind to I-RNA or deletion mutants thereof and thereformay be used (e.g., in binding assays) to identify other molecules havingthe translation inhibition activity of the I-RNA of the invention.UV-crosslinking studies utilizing various labeled RNAs and competitionexperiments demonstrated that both I-7 and I-4 mutant I-RNAs bound twocommon polypeptides namely 52 and 37 kDa (see Example 11). However,these two RNAs differed from each other in that I-7 RNA bound a 80 kDapolypeptide whereas I-4 RNA interacted with a 70 kDa polypeptide.Therefore, in addition to the 52 and 37 kDa polypeptides, binding of the80 kDa protein to the viral 5'-UTR may be important for internalinitiation to occur and I-7 RNA may directly compete with the 5'-UTR inbinding these polypeptides. A recent report by Meyer et al. utilizingUV-crosslinking studies indicates the importance of a 80 kDa protein inIRES-mediated translation of foot-and-mouth disease virus (FMDV) (Meyer,K., A. Petersen, M. Niepmann and E. Beck (1995) J. Virol. 69:2819-2824).This 80 kDa protein has been identified as initiation factor, eIF-4B.The results presented by Meyer et al. suggest that additional proteinfactors contribute to this interaction of eIF-4B with FMDV IRES.Therefore, binding of eIF-4B to viral IRES may require La and 37 kDapolypeptides and or other polypeptides. I-7 RNA may interfere withIRES-mediated translation by binding these polypeptides. Accordingly,ability to interfere ultimately with binding of the 80 kDa protein isexpected to be an indicator of an inhibitor having the translationinhibition activity of the I-RNA of the invention.

Despite their interactions with 52 and 37 kDa polypeptides I-4 and I-8RNAs may not efficiently inhibit translation because of their inabilityto interact with the 80 kDa polypeptide. Binding of the 70 kDa proteinto I-4 and I-8 RNA may inhibit their ability to interfere withIRES-mediated translation, perhaps by preventing these RNAs frominteracting with the 80 kDa polypeptide. Accordingly, lack of binding tothe 70 kDa protein also is expected to be an indicator of an inhibitorhaving the translation inhibition activity of the I-RNA of theinvention.

Identification of Other RNA Sequences for Inhibitory RNAOligonucleotides

It should be apparent from the above that various additional RNAsequences, besides that of the exemplary yeast I-RNA, may be used toderive additional translation inhibitory RNA oligonucleotides accordingto the invention. For instance, additional active oligonucleotides maybe derived from the complement ("antisense") of the sequence of theyeast I-RNA, since this complementary sequence also shows thetranslation inhibitory activity of the I-RNA sequence itself. See FIG.13. The same mutational and other analytical approaches described forthe I-RNA sequence are applied, with appropriate routine modifications.

In addition it is apparent that naturally occurring RNA sequences havinglittle or no sequence homology with the exemplary yeast I-RNA may bemodified as described herein to produce active RNA oligonucleotides ofthe invention. Thus, as discussed in the Background above, for instance,it is known that certain loops of the 5'UTR of various picornaviralRNAs, for instance, are responsible for binding of those mRNAs to theprotein factors require for IRES-mediated translation initiation.Indeed, the present disclosure shows by deletion analyses that the yeastI-RNA requires the mRNA to have internal ribosome entry site (IRES)sequences to inhibit translation of poliovirus RNA in vitro.Oligonucleotides containing these sequences also will have translationinhibitory activity according to this invention.

However, the prior art does not appear to have recognized thepossibility of using for translation inhibition according to thisinvention an RNA oligonucleotides consisting of the binding sequences ofsuch factor-binding loops, and even the production of such RNAoligonucleotides of less than 35 nucleotides, for any purpose, appearsto be unheralded.

Similarly, RNA loops shown to bind to other protein factors besides thep52 protein exemplified in this disclosure are also suitable sources ofnatural sequences for RNA oligonucleotides of the invention.Additionally, searches for sequences that are similar to I-RNA sequenceusing FASTA (Pearson et al. 1988) on Biovax copy of GCG-formattedGenbank of three databases, namely viral, structural RNA and plantsincluding yeast (version Sept. 1993) has identified partially relatedsequences. Thus, 89.5% homology was found (with a 19 nt overlap) with aregion of the Japanese encephalitis viral genome. Similarly, 70-80%homology was observed with different regions of genomes of herpessimplex, sindbis, Epstein-Barr, dengue and influenza viruses. Whencompared against the structural RNA database, the most striking homology(79-100%, 11-19 nt overlap) was with the 16S rRNA from a variety oforganisms. The I-RNA also has extensive homology (93.8%, 16 nt overlap)with 5S rRNA of trypanosomes. Comparison against the plant databasewhich includes yeast, showed 93.3% homology (15 nt overlap) with yeastgenes of histone H4.1 and H3. The only mRNA found to have homology withI-RNA was maize superoxide dismutase 3 isoenzyme (100% homology, 13 ntoverlap). Considering the short length of the overlapping sequences, thesignificance of these findings is unclear at present; however, thesesequences a preferred candidates to test for inhibitory activity of theinvention as described herein, particularly those which show appropriatepredicted secondary structure.

Identification of Structural Mimics of an Active RNA Oligonucleotide

To determine what secondary structure within the l-RNA molecule isimportant for its translation inhibitory activity, site-directedmutagenesis is performed by changing one or two nucleotides at a time todestabilize I-RNA secondary structures.

Two computer-predicated secondary structures of the I-RNA can beenvisioned that are relatively thermodynamically stable. Thesecomputer-predicted, secondary structures of I-RNA are confirmed bydetermining its (their) accessibility to modifications with dimethylsulfate (DMS) and sensitivity to single- and double-strand specificnucleases. These methods have been used to support computer-predictedsecondary structures of PV5'UTR.

Briefly I-RNA is treated with DMS, S1, cobra venom (CV), as well asother nucleases as described (11). For the identification of sites wheremodifications and cleavages have occurred, primer extension techniquewill be used (11). Elongation of DNA chains should stop one nucleotidebefore the modified base and just before the cleaved internucleotidebond. Initial deletion studies described earlier and experimentallyverified secondary structure of 1-RNA allow determination whichnucleotides to mutate within I-RNA. On finding mutations whichdestabilize secondary structures of I-RNA which also destroys itsbiological activity (both in vitro and in vivo), further compensatingmutations can be introduced to stabilize the secondary structure whichshould bring back its biological activity, thereby creating structuralmimics of an RNA oligonucleotide of the invention.

In addition structural mimics with enhanced resistance to nucleases orpermeability to membranes may be synthesized using convention nucleotideanalogs known in the art and testing for activity as described herein.

Further, a general method of obtaining an analog of a molecule thatbinds to an analyte is described in U.S. Pat. No. 5,133,866. Thisdescribes and claims method to identify a paralog useful for the conductof affinity chromatography with respect to an analyte which has specificaffinity for a first moiety in comparison to additional moieties presentin the environment of the first moiety which method comprises:screening, for ability to selectively bind said first moiety a panel ofindividual candidate paralogs, wherein said candidate paralogs havesystematically varied values of at least two different parameters, eachof which parameters determines the ability of the paralog to bind othersubstances. The method includes candidate paralogs which are nucleicacids. The method makes possible the systematic and facile selection ofa substance capable of specific binding to any selected moiety. If theselected moiety is a receptor or other biological target, the paralogwill be useful in a variety of pharmacological and therapeuticapplications.

Screening Assays for Inhibitors of the Invention

In vitro assays for identifying molecules which inhibit proteintranslation by the same mechanism as the yeast I-RNA of the invention,such as antibodies or other compounds that modulate the activity of aprotein translation initiation factor which binds to an internalribosome entry site and which is inhibited by binding to the yeast I-RNAof the invention, also are provided by the invention. Such assays mayinvolve an immobilized ligand, for example, immobilizing a suitableinitiation factor required for IRES-dependent protein translationinitiation or a molecular mimic of such a factor, or, alternatively, anatural ligand to which such an initiation factor binds (e.g., I-RNA),detectably labelling the nonimmobilized binding partner, incubating thebinding partners together and determining the effect of a test compoundon the amount of label bound wherein a reduction in the label bound inthe presence of the test compound compared to the amount of label boundin the absence of the test compound indicates that the test agent is aninhibitor of initiator factor binding.

Another type of assay for identifying compounds that modulate theinteraction between a translation initiation factor and a ligand is ascintillation proximity assay which involves immobilizing the factor ora mimic or fragment thereof on a solid support coated (or impregnatedwith) a fluorescent agent, labelling the ligand with a compound capableof exciting the fluorescent agent, contacting the immobilized initiationfactor with the labeled ligand in the presence and absence of a putativemodulator compound, detecting light emission by the fluorescent agent,and identifying modulating compounds as those compounds that affect theemission of light by the fluorescent agent in comparison to the emissionof light by the fluorescent agent in the absence of a modulatingcompound. Alternatively, the initiation factor ligand may be immobilizedand initiation factor may be labeled in the assay.

Yet another method contemplated by the invention for identifyingcompounds that modulate the interaction between an IRES-dependentprotein initiation factor and a ligand is a di-hybrid screening assay.This assay involves transforming or transfecting appropriate host cellswith a DNA construct comprising a reporter gene under the control of apromoter regulated by a transcription factor having a DNA-binding domainand an activating domain, expressing in the host cells a first hybridDNA sequence encoding a first fusion of part or all of a IRES-dependenttranslation initiation factor and either the DNA binding domain or theactivating domain of the transcription factor, expressing in the hostcells a second hybrid DNA sequence encoding part or all of the ligandand the DNA binding domain or activating domain of the transcriptionfactor which is not incorporated in the first fusion, evaluating theeffect of a putative modulating compound on the interaction betweeninitiation factor and the ligand by detecting binding of the ligand toinitiation factor in a particular host cell by measuring the productionof reporter gene product in the host cell in the presence or absence ofthe putative modulator, and identifying modulating compounds as thosecompounds altering production of the reported gene product in comparisonto production of the reporter gene product in the absence of themodulating compound. Presently preferred for use in the assay are thelexa promoter, the lexa DNA binding domain, the GAL4 transactivationdomain, the lacZ reporter gene, and a yeast host cell. Variations of thedi-hybrid assay may include interactions of La or p80 proteinsinteracting with other components of the translation apparatus.

A modified version of the foregoing assay may be used in isolating apolynucleotide encoding a protein that binds to a translation initiationfactor by transforming or transfecting appropriate host cells with a DNAconstruct comprising a reporter gene under the control of a promoterregulated by a transcription factor having a DNA-binding domain and anactivating domain, expressing in the host cells a first hybrid DNAsequence encoding a first fusion of part or all of the initiation factorand either the DNA binding domain or the activating domain of thetranscription factor, expressing in the host cells a library of secondhybrid DNA sequences encoding second fusions or part or all of putativeinitiation factor binding proteins or RNAs and the DNA binding domain oractivating domain of the transcription factor which is not incorporatedin the first fusion, detecting binding of an initiation factor bindingprotein or RNA to the initiation factor in a particular host cell bydetecting the production of reporter gene product in the host cell, andisolating second hybrid DNA sequences encoding initiation factor bindingprotein or RNA from the particular host cell.

An additional assay for a translation inhibitor having the activity ofthe I-RNA of the invention is an La-dependent in vitro translationassay. This approach is based on direct observation of inhibition ofIRES-mediated in vittro translation as described in the examples.Alternatively, compounds may be screened for inhibition ofIRES-dependent translation in transfected cells, for instance, using abicistronic RNA molecule with one protein product being translated in aCAP-dependent fashion and the second protein product translated viaIRES, as described in Example 10, for instance. Such a screen may bebased on inhibition of IRES-dependent translation of a reporter moleculefrom a cell culture. In addition, screening for inhibitors also mayutilize detection of inhibition of virus production (either capsidprotein as described in Example 10 or via plaque assay. Ultimately, ananimal-based screen for compounds that block production ofpicornavirus-mediated effects in an animal model system is used toevaluate efficacy of I-RNA mimics.

Use of the Inhibitor Molecules and Related Aspects of the Invention

The methods and inhibitor molecules of the invention may be used for thetreatment or prevention of viral infections in cells or in animal orhuman subjects. It also may be used as a diagnostic tool to determinewhether a particular RNA show IRES-mediated translation which generallyindicates a viral mNRA.

As to the range of viruses suitable for the invention, the inhibitoryRNA from yeast specifically inhibits IRES-mediated translation by avariety of picornaviral RNAs including those of poliovirus, rhinovirus,hepatitis A virus, coxsackievirus, and other members of thepicornaviridae group. Translation of capped cellular mRNAs does notappear to be affected by this yeast inhibitor RNA in vitro or in vivo,whereas picornaviral replication is inhibited by the yeast inhibitor RNAin vivo due to inhibition of viral RNA translation. The inhibitor RNAspecifically binds proteins which interact with RNA structural elementswithin the viral 5'UTR.

Many other viruses not belonging to the picornaviral group also useinternal ribosome entry for translation. A prime example is aflavivirus, hepatitis C (1, 2). The yeast inhibitor also inhibitshepatitis C virus translation. Recently, it has been reported that mRNA3 of infectious bronchitis virus, a coronavirus, also utilizes internalribosome entry mechanism (3, 4). In addition, mRNAs encoding reversetranscriptase of duck and human hepatitis B virus, vesicular stomatitisvirus NS protein, adenovirus DNA polymerase, and Sendai virus P/Cprotein have been shown to use internal initiation of ribosome entry(5-9). Further still, internal ribosome entry has been shown fortranslation in the retrovirus family (e.g., murine leukemia virus; ref.29), the pestivirus family (30), and plant poty viruses (31). Thus,antiviral agents against members of many different virus groups whichutilize internal initiation of translation may be prepared according tothe present invention.

The inhibitory RNAs and structural mimics of the invention can also beused to control the translation of an internally initiated mRNA, such asa viral mRNA, in a cell culture or host organism harboring such a mRNA.The inhibitory RNA or mimic is supplied using standard methods ofadministration, such as those set forth in Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa., latest edition.Preferably, for in vivo treatment of a subject, the RNA or mimic isprovided by injection, and formulated using conventional excipientstherefor, such as Ringer's solution, Hank's solution, and the like. Oraladministration with proper formulation can also be effected. While mostadministration is systemic, in the case of localized conditions such asa nasal infection by rhinovirus, administration may be topical orotherwise local. Slow release mechanisms for drug delivery may also beused.

Alternatively, the inhibitory RNA sequence may be generated in situ byproviding an expression system which scontains a DNA encoding the RNA orinhibitory effective fragment thereof in a "reverse orientation"expression system. The expression system may either be designed to beoperable in the host subject, such as a mammalian subject wherein thereverse oriented sequence is under the control of, for example, an SV-40promoter, an adenovirus promoter, a vaccinia virus promoter or the like,so that the RNA is transcribed in situ. When used in a culture of hostcells, the expression system will be provided on a replicon compatiblewith the host cells.

More in particular, the yeast I-RNA of the invention has been shown toinhibit IRES-dependent initiation of translation from the 5'UTR ofpicornaviruses including human picornaviruses (poliovirus, rhinoviruses,hepatitis A and coxsackievirus B3) and an animal picornavirus(foot-and-mouth virus; FMDV), as well as internal translation of aflavivirus (hepatitis C) mRNA. Further, mRNA 3 of infectious bronchitisvirus, a coronavirus which causes significant losses in the poultryindustry, also utilizes an internal ribosome entry mechanism (3, 4). Inaddition, mRNAs encoding reverse transcriptase of duck as well as humanhepatitis B virus, vesicular stomatitis virus NS protein, adenovirus DNApolymerase, and Sendai virus P/C protein have been shown to use internalinitiation of ribosome entry (5-9). Further still, internal ribosomeentry has been shown for translation in the retrovirus family (e.g.,murine leukemia virus; ref. 29), the pestivirus family (30), and plantpoty viruses (31). Accordingly, this invention also enables productionof transgenic animals and transgenic plants, using available geneticengineering technology, which express an I-RNA molecule or relatedtranslation initiation inhibitor of the invention and thereby areresistant to pathogenic effects of viruses in which the I-RNA inhibitsIRES-dependent translation. We contemplate the production of transgenicplants and animals, by conventional techniques, that are resistant toviruses and other pathogens whose replication depends upon internalinitiation of translation.

Another aspect of the invention relates to isolation and modification ofI-RNA genes in cells, particularly in yeast cells, which express anI-RNA of the invention that prevents IRES-dependent translationinitiation of a desired mRNA in such cells or extracts thereof. Thesegenetic modifications inhibit expression or activity of the I-RNA,thereby allowing the desired IRES-dependent translation initiation. Inthe first instance, identification of the sequence of the I-RNA moleculeisolated from yeast, as described herein, enables preparation of alabeled probe which can be used with conventional genetic engineeringmethods detect the I-RNA gene (e.g., by Southern blotting) and itsinitial transcription product (e.g., by Nothern blotting). Further,using such a probe, preferably under stringent hybridization conditions,the yeast I-RNA gene or homologous genes from other species ortranscripts of such genes may be isolated by standard gene cloningapproaches well known in the art. For example, a random genomic libraryof a desired species may be screened by hybridization using an I-RNAprobe provided by the invention. Examination of the structure andgenomic organization of the yeast I-RNA gene and homologous genes fromother species will provide a better understanding of the normal functionof such genes.

In addition, the present disclosure enables modifications of host cellsto inhibit expression or activity of I-RNA. In the first instance,introduction of such modifications will determine whether I-RNA activityis essential for survival of host cells which express such I-RNAs. Inone approach, an RNA having a sequence complementary to the I-RNA (i.e.,an "antisense I-RNA") is expressed in a host cell (e.g., yeast) whichexpresses an I-RNA molecule. The vector for expression of the antisenseI-RNA contains a selectable marker gene (e.g., URA 3) to ensure thatonly transformed cells are recovered. If no transformed cells expressingantisense I-RNA are recovered, inducible expression constructs may betested to determine whether the antisense RNA vector can be transformedinto the cell in absence of antisense I-RNA expression and whethersubsequent expression inhibits any cellular function(s). Alternatively,the I-RNA gene may be eliminated using gene "knock out" methodologyknown in the art. For instance, in yeast cells exogenous DNA introducedinto the cell efficiently and stably integrates into chromosomal DNA byhomologous recombination, allowing efficient replacement of a wildtypegene with a non-functional copy. Typically, the non-functional copy isgenerated by replacing wildtype coding sequences with a selectablemarker gene (e.g., LEU or URA). Transformation of diploid cells maycircumvent possible lethal effects if some I-RNA activity is requiredfor cell viability. Yeast or other I-RNA-expressing host cells, orextracts thereof, which have reduced I-RNA activity as a result ofeither an antisense or gene knock out modification according to theinvention, are useful for expression of mRNAs requiring IRES-dependenttranslation initiation. Also contemplated are yeast or other I-RNA hostcells which can be modified by gene knockout methodologies, as known inthe art, to remove the gene encoding La or homologs thereof to producehost strains that are permissive for expression of proteins whosesynthesis is dependent upon internal initiation of translation.

The following examples are intended to illustrate, but not to limit, theinvention.

EXAMPLE 1 Purification and Sequencing of the Yeast Inhibitor RNA

The Saccharomyces cerevisiae strain used was ABYSI (provided by D.Meyer, UCLA). The inhibitor from yeast cells capable of specificallyinhibiting IRES-dependent translation from poliovirus RNA was initiallypurified by passage through a DEAE-Sephacel column (Coward et al. 1992,supra). The inhibitor bound strongly to the column and was eluted bywashing the column with 1M potassium acetate. Further purification ofthe DEAE-Sephacel purified inhibitor involved DNase and proteinase Kdigestion followed by phenol-chloroform extraction. Finally, RNAobtained by alcohol precipitation of the aqueous phase was end-labeledwith γ³² P-ATP at the 5'-end, and single RNA bands were resolved by 20%PAGE/8M urea electrophoresis. Each RNA band was eluted from gel slicesand was assayed for its ability to inhibit internal initiation oftranslation from a poliovirus 5'UTR-CAT construct but not from a controlCAT construct known to initiate translation in a cap-dependent manner(Pelletier et al. 1989). A single band which migrated as an RNA of 60nucleotides was associated with the inhibitory activity.

More in particular, yeast cell lysates were prepared as describedpreviously (Rothblatt et al. 1986) except that they were not treatedwith micrococcal nuclease. Lysates were loaded onto a DEAE Sephacel(Pharmacia) column at 0.1M potassium acetate and step-eluted withbuffers containing 0.3, 0.6 and 1M potassium acetate. The fractions weredialyzed back to 0.1M salt and assayed for translation inhibitoryactivity. The 1M fraction which showed inhibitory activity was subjectedto DNase treatment followed by proteinase K digestion andphenol-chloroform extraction. The RNA from this fraction was thenisolated by alcohol precipitation. The yeast RNAs that copurified withthe 1M fraction were then dephosphorylated and 5' end labeled by kinasereaction. Labeled RNA species were separated on a 20% acrylamide-8M ureasequencing gel. Labeled and cold RNA bands were run in parallel lanesand were eluted from the gel as follows. Individual gel slices weresoaked in 500 μl of elution buffer (2M ammonium acetate and 1% SDS) at37° C. for 4 hr. After a brief centrifugation at room temperature thesupernatant was collected, extracted with phenol:chloroform (1:1), andalcohol precipitated in the presence of 20 μg of glycogen(Boehringer-Mannheim Biochemicals).

The precipitated RNA pellets were resuspended in nuclease-free water andtested for the ability to inhibit translation of p2CAT RNA (Coward etal. 1992, supra) in the HeLa cell-free translation system. HeLa cellswere grown in spinner culture in minimal essential medium (GIBCOlaboratories) supplemented with 1 g/L glucose and 6% newborn calf serum.HeLa cell extracts were prepared as previously described (Rose et al.1992; Coward et al. 1992, supra). In vitro translation in HeLa cell-freeextracts was performed essentially as described earlier (Rose et al.1978). Two micrograms of each mRNA were used with 80 μg of HeLa cellextract in a 25μl reaction mixture in presence of 25 μCi of ³⁵S-methionine (800 Ci/mmol; Amersham) and 40 units of RNasin (Promega).

Translation in rabbit reticulocyte lysate (Promega) was performed in 15μl reaction volumes that contained 12.5 μl of the lysates, 2 μg of mNRAwith 25 μCi of ³⁵ S-methionine (specific activity>1000 Ci/mmol) at 30° Cfor 1 hr. Three microliters of the translation product was analyzed bysodium-dodecyl sulfate (SDS) - 14% polyacrylamide gel electrophoresis.

The purified RNA was sequenced using a commercially available sequencingkit (US Biochemicals Corporation). The end-labeled RNA was mixed withbase specific ribonuclease-buffer combination (following theUSB-protocol), incubated at 50° C and then loaded onto a 20%acrylamide-SM urea sequencing gel. FIG. 1A shows the sequence [SEQ IDNO:1] of the 60 nucleotide RNA.

EXAMPLE 2 Cloning and Transcription of the Yeast Inhibitor RNA

Based on the determined RNA sequence, sense- and antisense-strandspecific deoxyoligonucleotides were synthesized, annealed and clonedinto the pGEM 3Z expression vector between the HindIII and EcoRI sitesin the polylinker region to form the recombinant plasmid pSDIR (FIG.1B).

The clone pSDIR was linearized with HindIII restriction enzyme, thentranscribed with the T7 RNA polymerase to generate the inhibitor RNA(sense transcript). Transcription by T7 RNA polymerase from thelinearized plasmid resulted in synthesis of the inhibitor RNA. Whenanalyzed by gel electrophoresis a single band of 71 nucleotides wasobserved, comprising the yeast inhibitor RNA and an extra tennucleotides from the EcoRI site from the 5'-polylinker region, and onenucleotide at the 3'-end from the HindIII site.

To determine whether the RNA synthesized from the synthetic clone wasactive, its effect on translation from a CAT construct containingpoliovirus 5'UTR at the 5'-end of the CAT gene (P2-CAT) was determined.Both the partially purified inhibitor from yeast and the purifiedinhibitor transcribed from pSDIR inhibited translation from the P2CATRNA in vitro in a HeLa cell-free extract (Figure 1D, lanes 4, 5, 6).However, translation from CAT RNA (cap-dependent translation) was notsignificantly inhibited by either inhibitor (FIG. 1D, lanes 1, 2, 3).Thus, the inhibitor RNA synthesized from the synthetic clone was activein specifically inhibiting poliovirus IRES-dependent translation aspreviously found with the partially purified inhibitor from yeast cells(Coward et al. 1992, supra).

EXAMPLE 3 Identification of Poliovirus 5'UTR Sequences Required forInhibition by the Yeast RNA Inhibitor

To determine whether specific sequences within the 5'-untranslatedregion (UTR) of poliovirus RNA are required for the yeast inhibitor RNAto inhibit IRES-dependent translation, a number of deleted 5'UTR-CATconstructs were obtained from the laboratory of Dr. N. Sonenberg (McGillUniversity). See FIG. 2D.

The mRNAs were transcribed in vitro using either T7 or SP6 promoter fromdifferent linear plasmids by T7 or SP6 RNA polymerases. Both plasmidpG3CAT and P2CAT (Coward, et al. 1992, supra) were linearized with BamHIand the runoff transcripts were generated using SP6 RNA polymerases. Theplasmid pBIP-LUC construct was obtained from P. Sarnow (Macejak et al.1991) and was linearized with HpaI enzyme and transcribed with T7 RNApolymerase. The TMEV-IRES containing construct pPB310 was from Howard L.Lipton (Bandopadhyay et al. 1992) and was linearized with HpaI andtranscribed with T7 RNA polymerase.

Oligodeoxyribonucleotide templates for transcription by T7 RNApolymerase were synthesized on an Applied Biosynthesis DNA synthesizerand then purified. Equimolar amounts of the 18 mer T7 primeroligonucleotide and the template oligonucleotides were mixed in 0.1MNaCl and were annealed by heating at 100° C. for 5 min followed by slowcooling to room temperature. The SL-B, SL-C, SL-D, and SL-G, RNAs weresynthesized in vitro following the method described above.

The inhibitor efficiently inhibited translation from pP2 CAT but notfrom the pG3 CAT (or pCAT) construct (FIG. 1D). Almost completeinhibition was observed when the Δ5'-33 CAT construct was translated inthe presence of the inhibitor RNA (data not shown). Deletion of thefirst 320 nucleotides from the 5'-end of the UTR did not have asignificant effect on the ability of the inhibitor to inhibittranslation from the A5'-320/CAT construct (FIG. 2A, lanes 1 and 2).Almost 80% inhibition of translation was observed in the presence of theinhibitor (lanes 1 and 2). Some of the inhibition observed with theinhibitor could be reversed when the template RNA was capped prior totranslation (FIG. 2A, lanes 3 and 4).

A similar result was obtained with the Δ3'-631/CAT construct; almostcomplete inhibition of translation from this construct was observed inthe presence of the inhibitor and addition of capped RNA reversedinhibition of translation to some extent but not as much as that withA5'-320 construct (FIG. 2B, lanes 5-8). In contrast, translation fromthe Δ5'-632/CAT construct was almost unaffected in the presence of theinhibitor (FIG. 2B, lanes 1 and 2).

Therefore, almost the entire IRES region of viral RNA (320-631) isrequired for the inhibitor to efficiently inhibit almost all poliovirusIRES-dependent translation. However, significant inhibition was observedwith a construct containing only nucleotides 320-461 of the viral UTR.Thus, translation from the construct Δ5'-320/Δ3'-461/CAT containing onlynucleotides 320-461 of the UTR was significantly inhibited by theinhibitor RNA (FIG. 2C, lanes 1 and 2). This inhibition can be overcometo a significant extent by capping the RNA prior to translation (FIG.2C, lanes 3 and 4), indicating that cap-dependent translation issubstantially unaffected by the inhibitor.

EXAMPLE 4 Demonstration of Disruption of Complexes of Protein Factorsand mRNA 5'UTR Sequences by Yeast I-RNA using RNA Retardation during GelElectrophoresis

Theoretically, the inhibitor RNA could inhibit IRES-dependenttranslation by two possible mechanisms: binding to UTR sequences as anantisense RNA or binding to protein factors needed for internal entry ofribosomes. To distinguish these two mechanisms, uniformly ³² P-labeledinhibitor RNA probe was prepared and mixed with HeLa S10 extracts, andthe resulting RNA-protein complexes were analyzed by nondenaturingpolyacrylamide gel electrophoresis.

HeLa S10 cytoplasmic extract was prepared by collecting the supernatantafter centrifugation of the HeLa cell free translation extract at 10,000g, for 30 min. at 4° C. A 50 μg sample of S10 extract was preincubatedat 30° C. for 10 min with 4 μg of poly [d(I-C)] (Pharmacia) in a 15 μlreaction mixture containing 5 mM HEPES pH 7.5, 25 mM KCl, 2 mM MgCl₂,1.0 mM EDTA, 3.8% Glycerol and 2 mM DTT. For competition experiments10-100-fold excess of the unlabeled competitor RNAs were added to thereaction and incubated for 10 min at 30° C. Finally 5-10 fmole of thelabeled RNA probes were added to the respective reaction mixtures andthe incubation was continued for another 30 min at 30° C. Thenonspecific RNA used in the competition assays was the sequence of thepolylinker region (EcoRI to HindIII) of the pGem3Z vector (Promega).Three microliters of the gel loaded dye were added to the reactionmixture to a final concentration of 10% glycerol and 0.2% of bothbromophenol blue and xylene cyanol. The RNA-protein complexes were thenanalyzed on a 4% polyacrylamide gel (39:1-acrylamide:bis) in 0.5X TBE.

As shown in FIG. 3A, a single complex (denoted C) was clearly evident.Increasing concentrations of unlabeled I-RNA competed with the formationof the labeled complex (FIG. 3B, lanes 2-5). A similar result wasobtained when unlabeled poliovirus 5'UTR RNA was used for competition.Clearly, at the highest concentration tested UTR sequences successfullycompeted with I-RNA for binding to HeLa S10 proteins (FIG. 3B, lanes6-9). An unrelated RNA, however, was not able to compete with labeledI-RNA (FIG. 3B, lane 10). Thus, the inhibitor RNA was able to form agel-retarded complex with HeLa S10 protein(s) that can be specificallycompeted with viral 5'UTR sequences.

EXAMPLE 5 Demonstration that the Inhibitor RNA Binds Proteins thatInteract with Poliovirus 5'UTR

To determine whether specific polypeptides that interact with the viral5'UTR also interact with the yeast inhibitor RNA, a series ofUV-crosslinking experiments were performed. In these experiments theuniformly labeled inhibitor RNA was first incubated with a HeLa S10extract and then crosslinked using UV light. After ribonucleasetreatment, protein-nucleotide complexes were analyzed bySDS-polyacrylamide gel electrophoresis.

Forty to fifty fmole of the ³² P-labeled RNA probes (8×10⁴ cpm) wereincubated with 50-100 μg of S-10 extract of HeLa cells as describedabove. After the binding reaction was complete, the samples wereirradiated with UV light from a UV lamp (multiband UV-254/366NM Model UGL-25; UVP, Inc.) at a distance of 3-4 cm for 10 min. The unbound RNAswere digested with a mixture of 20 μg of RNase A and 10U of RNase T1 at37° C. for 30 min and then analyzed on SDS-14% polyacrylamide gels.

When ³² P-labeled inhibitor RNA was used to crosslink proteins in HeLaextract, polypeptides having approximate molecular weights of 100, 70,52, and 37 kDa were detected (FIG. 4A, and 4B lane 2). Among thesepolypeptides, the 52 kDa protein was most intensely labeled in someexperiments (FIG. 4B, lane 2). Addition of unlabeled inhibitor RNAsuccessfully competed with the 52 kDa band (FIG. 4A, lane 3). Whenunlabeled poliovirus 5'UTR was used as a competitor, the 52 kDa as wellas the 100, 70 and 37 kDa bands were completely competed out (FIG. 4A,lane 4). In contrast an unlabeled, unrelated RNA was unable to competewith any of the polypeptides crosslinked to the inhibitor RNA (lane 5).

Because a 52 kDa protein has previously been shown to interact with aspecific region of the viral 5'UTR (nucleotides 559-624), the ability ofan RNA containing this sequence ("UTR 559-624") to compete with the 52kDa band crosslinked to labeled I-RNA was determined. As shown in FIG.4B (lane 4), unlabeled UTR 559-624 completely inhibited formation of theI-RNA-52 kDa complex. Unlike the whole 5'UTR sequence, UTR 559-624competed with only the 52 kDa protein (compare lane 4 of FIGS. 4A and4B). These results indicate that the yeast inhibitor RNA binds a 52 kDaprotein similar or identical to that bound by poliovirus 5'UTR sequence559-624.

EXAMPLE 6 Demonstration that the Yeast Inhibitor RNA Competes with BothStem-loops D and G for Protein Binding

Poliovirus 5'UTR contains several thermodynamically stable stem-loopstructures that are believed to play important roles in viral RNAreplication and translation (FIG. 5). Among these, stem-loops A, B and Care presumably involved in RNA replication. On the other hand stem-loopsD-G are believed to be involved in viral mRNA translation (Dildine etal. 1992). Stem-loop D (SL-D), comprising nucleotides 186-221, has beenshown to bind a 50 kDa protein (p50) (Najita et al. (1990, supra)),whereas stem-loop G (SL-G), representing poliovirus 5'UTR sequences from559-624, binds to a 52 kDa protein (p52) which has recently beenidentified as the human La protein (Meerovitch et al. (1993, supra)).

Results presented in the previous Example indicated that the inhibitorRNA interacts with p52 which normally binds to stem-loop G within theviral 5'UTR. To determine if the inhibitor RNA is also capable ofbinding to p50 and competing with stem-loop D, an RNA corresponding tonucleotides 178-224 of the 5'UTR was prepared. In the first experimentindividually ³² P-labeled I-RNA, the whole 5'UTR, stem-loop G (UTR559-624), and stem-loop D (UTR 178-224) were incubated separately withHeLa S-10 extract, and the resulting protein-nucleotide complexes wereanalyzed by UV-crosslinking as shown in FIG. 6A. A majorprotein-nucleotidyl complex of approximately 52 kDa was detected in allfour reactions (lanes 2, 4, 6, and 8).

When stem-loop G was used as the labeled probe, an additional band at 54kDa was detected (FIG. 6A, lane 6). Other crosslinked proteins rangingfrom 37 to 48 kDa were also detected with all four labeled probes.Because the lane showing protein binding by labeled stem-loop G (FIG.6A, lane 6) was relatively overexposed, another experiment comparingbinding of SL-G and SL-D was performed (FIG. 6B, lanes 1 and 2). Thisclearly shows that both stem-loops D and G bind proteins which migrateidentically on the SDS-gel (52 kDa band).

To confirm the result that similar proteins may be interacting withI-RNA and stem-loops D and G, the following competition experiments wereperformed. ³² P-labeled 5'UTR or SL-D (UTR 178-224) or SL-G (UTR559-624) RNAs were incubated with HeLa S10 extract either alone or inthe presence of unlabeled competing RNAs (e.g., 5'UTR, SL-D, I-RNA,SL-B, SL-C, nonspecific RNA). The resulting complexes were then analyzedby UV-crosslinking studies. RNA sequences representing stem-loops B andC (SL-B and SL-C) were used as negative controls.

When poliovirus 5'UTR was used as the labeled probe, almost completeinhibition of formation of the 52 kDa protein-nucleotidyl complex wasobserved with unlabeled UTR, I-RNA and stem-loop G (FIG. 7A, lanes 2-5).Stem-loop D partially competed with labeled 5'UTR for p52 binding (FIG.7A, lane 6). Unlabeled SL-B, SL-C and a nonspecific RNA were relativelyineffective in competing with 5'UTR for p52 binding (FIG. 7A, lanes7-9). Only unlabeled UTR RNA competed with the formation of all labeledbands whereas I-RNA and stem-loops G and D specifically inhibitedformation of the p52 band.

When stem-loop G was used as the labeled probe, a doublet migrating at52 and 54 kDa was detected as expected (FIG. 7B, lane 2). Homologouscompetition with unlabeled SL-G completely inhibited formation of thesecomplexes (FIG. 7B, lane 3). Almost 80% inhibition of formation of theseUV-crosslinked complexes was observed in the presence of unlabeled I-RNAand SL-D RNA (lanes 4, 5). However, no inhibition was observed when anunrelated RNA and SL-B or SL-C RNAs were used as competitors (lanes6-8). A similar result was obtained when radiolabeled SL-D RNA was usedas the probe in the UV-crosslinking assay. Almost total inhibition ofthe formation of protein-nucleotidyl complex was observed in thepresence of unlabeled I-RNA, SL-G RNA and the homologous SL-D RNA (FIG.7C, lanes 2-4 and 6). As expected SL-B and SL-C RNAs were unable tosuccessfully compete with the labeled SL-D probe.

Taken together these results show that a similar or identical proteinhaving an approximate molecular weight of 52 kDa interacts with allthree RNAs: stem-loops D and G, and I-RNA.

EXAMPLE 7 Demonstration that the Yeast Inhibitor RNA PreferentiallyInhibits Internal Initiation of Translation In Vitro

To examine whether the cloned and purified I-RNA preferentially inhibitsinternal initiation of translation from an RNA also initiated from a 5'cap, its effect on translation from a bicistronic messenger wasdetermined. For this purpose, a bicistronic construct containing CAT andluciferase (LUC) genes flanked by the Thieler's Murine encephalomyelitisvirus (TMEV) 5'UTR was obtained. TMEV 5'UTR is known to contain IRESsequences and has been shown to initiate translation internally(Bandopadhyay et al. 1992). Initiation of translation occurringinternally from the TMEV 5'UTR would result in synthesis of luciferase,whereas cap-dependent translation would normally produce the CATprotein.

When translated in reticulocyte lysate, translation from the bicistronicmessage produced both CAT and luciferase proteins (FIG. 8B, lane 1). Inthe presence of the purified yeast inhibitor RNA, significant CATsynthesis was observed whereas synthesis of luciferase was almostcompletely inhibited. Quantitation of the labeled CAT and luciferasebands showed that luciferase synthesis was inhibited over 90% comparedto the control, while only 20% inhibition of CAT synthesis was observed.Background incorporation in the translation reaction also wassignificantly less in the reaction containing the inhibitor, for reasonsnot completely understood.

Using bicistronic RNA constructs it has been demonstrated that I-RNApreferentially inhibits internal ribosome entry site (IRES) mediatedtranslation by a variety of picornaviral RNAs including those ofpoliovirus, rhinovirus, hepatitis A virus, TMEV virus, and the like.See, for instance, FIG. 11.

These results indicate that the yeast inhibitor RNA preferentiallyinhibits internal initiation of translation in a second viral controlregion, the TMEV 5'UTR.

EXAMPLE 8 Demonstration that the Yeast Inhibitor RNA Inhibits InternalInitiation of a Cellular mRNA

Recent results have shown that some cellular mRNAs initiate translationinternally (Macejak et al. 1991; Oh et al. 1992). For instance, theimmunoglobulin heavy chain binding protein (Bip) can be synthesized byinternal initiation. To determine whether Bip synthesis can bespecifically inhibited by the inhibitor RNA, a construct containing the5'UTR of Bip mRNA linked to a reporter gene (luciferase) was obtainedfrom P. Sarnow (Univ. of Colorado). Translation of this mRNA in HeLaextracts generated the luciferase protein (FIG. 8A, lane 1). Addition ofthe yeast inhibitor RNA completely inhibited luciferase synthesis fromthis RNA construct (lane 2). As expected, cap-dependent translation froma CAT construct was not at all inhibited under the same conditions(lanes 3 and 4). In fact, CAT translation was significantly stimulatedover the control as previously observed (Coward et al. 1992, supra).These results indicate that the yeast inhibitor RNA was capable ofinhibiting internal initiation from a cellular mRNA as well as a viralmRNA.

EXAMPLE 9 Demonstration of Inhibition of Translation of Poliovirus RNAin vivo

To determine whether the cloned inhibitor RNA inhibits translation ofpoliovirus RNA in vivo, poliovirus RNA was transfected into HeLa cellsalone or together with the purified yeast RNA. HeLa cell monolayers weregrown in tissue culture flask in minimal essential medium (GIBCO)supplemented with 5% fetal bovine serum. Poliovirus RNA (type 1 Mahoney)was isolated from infected HeLa cells as described earlier (Dasgupta,1983). Synthetic I-RNA or polio RNA were mixed with carrier yeast tRNAto yield a total 20 μg RNA per transfection reaction. The RNA sampleswere then mixed with 30 μg of Lipofectin (GIBCO-REL) and 20 units ofRNasin (Promega) and incubated for 30 min at room temperature. Finally,the samples were mixed with 4 ml of minimal essential medium (GIBCO)containing 2.5% fetal bovine serum, and added to petri dishes containing70-80% confluent HeLa monolayer cells. The cells were then incubated at37° C. in a CO₂ incubator for 24 hr.

Proteins were labeled by addition of ³⁵ S-methionine and synthesis ofviral proteins was monitored by direct analysis of cell-free extracts(FIG. 9, panel A) or by immunoprecipitation of viral capsid proteins byanticapsid antisera (FIG. 9, panel B). More in particular, for in vivolabeling of proteins after transfection, cells were preincubated inmethionine-free medium (MEM, GIBCO) for 40 min at 37° C. Then 100 μCi ofthe trans labeled methionine (sp. act.>1000 Ci/mmole) was added to thecells and incubation was continued for another hour.35S-methionine-labeled HeLa cell extract was prepared as describedpreviously (Ransone et al. 1987).

In vivo labeled viral proteins in transfected cells were detected byimmunoprecipitation with poliovirus anticapsid antibody (purchased fromAmerican Type Culture Collection). Immunoprecipitations were performedovernight at 40° C. with 5 μl of anticapsid antibody in a 500 μlreaction volume containing IxRIPA buffer (5 mM Tris pH 7.9, 150 mM NaCl,1% Triton X100, 0.1% SDS, 1% Sodium deoxycholate). The immune complexeswere precipitated with protein A Sepharose (75 μl of a 20% solution inRIPA buffer plus 0.2% BSA) and then analyzed on a SDS-14% polyacrylamidegel as described earlier (Coward et al. 1992, supra) .

In the absence of added viral RNA and the inhibitor RNA, synthesis ofcellular proteins was evident (panel A, lane 1). When viral RNA (1 μg)was transfected into cells, synthesis of distinct viral proteins wasobserved (lane 2). In addition, the background of host cell proteinsdiminished considerably due to shut-off of host cell protein synthesisby poliovirus (lane 2).

When the inhibitor RNA was cotransfected with viral RNA (1 μg) into HeLacells, no detectable synthesis of viral proteins was observed and hostcell protein synthesis was restored (lane 3). Expression of theinhibitor RNA alone did not interfere with the synthesis of cellularproteins (lane 4). Lane 5 (panel A) shows that carrier tRNA used intransfection experiments had no effect on cellular protein synthesis.

Transfection of cells with an increased amount of poliovirus RNA (2 μg)resulted in synthesis of viral proteins and a more pronounced shut-offof cellular protein synthesis (lane 6). However, in the presence of theinhibitor RNA, viral protein synthesis was inhibited and host cellprotein synthesis was restored (lane 7) to the level seen in the controlreaction. The results shown in panel B confirmed the fact that viralprotein synthesis was inhibited in cells containing the inhibitor RNA.Synthesis of viral capsid proteins was inhibited in cells cotransfectedwith viral RNA and the inhibitor RNA (lane 3, panel B).

Therefore, the yeast inhibitor RNA efficiently inhibited translation ofpoliovirus RNA in vivo. Further, protection of monolayer cells from thecytolytic effects of poliovirus infection in the presence of the yeastinhibitor RNA paralleled restoration of host cell protein synthesis seenin lanes 3 and 7 (FIG. 9A).

EXAMPLE 10 Analysis of Deletion Mutants of Yeast I-RNA

To determine I-RNA sequences required for inhibition of poliovirusIRES-mediated translation, a nested set of 15 nt long deletions of theyeast I-RNA sequence was generated (designated I-l, I-2, I-3, and I-4;FIG. 16). I-RNA deletion mutants were generated by in vitrotranscription with T7 RNA polymerase from oligonucleotide templates.Different lengths of oligonucleotides were synthesized (BiosynthesisInc.) each beginning with a T7 promoter adapter sequences followed byvarious lengths from different regions of I-RNA sequences.ligodeoxyribonucleotide templates were mixed with equimolar amounts ofthe 17 mer T7 primer oligonucleotide in 0.1M NaCl, and annealed byheating at 100° C. for 5 min followed by slow cooling to roomtemperature. The nucleotide positions of the different I-RNA deletionmutants are shown in FIG. 16.

Effects of truncated I-RNA mutants on in vitro translation programmed byP2 CAT RNA containing the poliovirus 5 UTR were determined. Both I-1 andI-2 RNAs were still active in translation-inhibition, although they werenot as active as intact I-RNA. Deletion of nucleotides 31-45 or 46-60(I-3 or I-4) from the I-RNA, however, almost totally destroyed theability to inhibit IRES-mediated translation, as shown by inhibition ofin vitro translation in HeLa lysates programmed by the 5'-UTR ofpoliovirus RNA. In particular, the effects of different I-RNA deletionmutants on in vitro translation of pG3CAT and P2CAT RNAs in HeLa lysateswere determined. in vitro translations were performed with approximately2 pg of either uncapped p2CAT RNA or capped pG3CAT RNA in the absence orpresence of the 2 pg of deleted I-RNAs.

The results of these tests indicated that the 3'-terminal half of theI-RNA contains major sequences necessary for inhibition of viralIRES-mediated translation. However, sequences present within the first15 nucleotides (i.e., deleted in I-1) or the next 15 nucleotides (I-2)also play a role in inhibition, since mutants lacking these sequencesare not as active as intact I-RNA. Further deletion analysis showed thata 25 nt long fragment of I-RNA (1-7 RNA, nt 26-50) was as active asI-RNA in viral translation-inhibition. A similar deletion mutant thatcontained an extra 10 nucleotides at its 3'-end (I-6 RNA, nt 26-60) alsowas active, but not as active as I-7 RNA. A fragment of I-RNA containingnt 1-25 (I-8 RNA), however, was totally inactive in translationinhibition. Further deletion of I-7 RNA resulted in a smaller fragment(I-9, nt 30-45) which was capable of inhibiting IRES-mediatedtranslation. The ability of this fragment (I-9) to inhibit translationis fully consistent with the previously noted inability of I-3 RNA toarrest translation since I-3 RNA lacks nts 31-45 (FIG. 3).

None of the tested mutant I-RNAs was able to inhibit cap-dependenttranslation of pCAT RNA.

To determine whether the truncated I-RNAs are capable of inhibitinginternal initiation of translation, their effects on translation from abicistronic construct were determined. A bicistronic constructcontaining CAT and luciferase (LUC) genes flanked by the poliovirus5'UTR was used in this experiment. Initiation of cap-independenttranslation occurring internally from the poliovirus 5'UTR would resultin the synthesis of luciferase, whereas cap-dependent translation wouldnormally produce the CAT protein. In uninfected HeLa cell extract,translation from the capped bicistronic message produced both CAT andluciferase proteins in separate experiments using different cellextracts. Addition of full length I-RNA preferentially inhibitedsynthesis of luciferase but not that of CAT. The 25 nt long I-7 RNAalmost totally inhibited production of luciferase. CAT synthesis,however, was significantly stimulated in reactions containing I-7 RNA.

No inhibition of luciferase synthesis was apparent with the mutant I-4RNA. Synthesis of CAT protein was also stimulated by I-4 RNA. Similarresults were obtained with I-9 and I-8 RNAs. The 16 nt long I-9 RNAinhibited luciferase synthesis significantly compared to the control.Although 20% inhibition of CAT production was observed in presence ofI-9 RNA, luciferase synthesis was inhibited almost 85% over the control.In contrast, I-8 RNA did not significantly inhibit synthesis of eitherluciferase or CAT. These results suggest that I-7 and I-9 RNAs, but notI-4 and I-8 RNAs, preferentially inhibit internal initiation oftranslation programmed by poliovirus 5 UTR.

To determine whether mutant I-RNAs inhibit translation of poliovirus RNAin vivo, poliovirus RNA was transfected (using liposomes) into HeLacells singly or together with purified I-7, I-9, I-4, I-8 and I-RNA.Proteins were labeled by ³⁵ S-methionine and the synthesis of viralproteins was monitored by immunoprecipitation of viral capsid proteinsfrom cell extracts by anti-capsid antisera. No capsid protein could beprecipitated from mock-transfected cells. Upon transfection of cellswith poliovirus RNA alone, synthesis of capsid proteins was clearlydetected. Cotransfection of I-7 RNA or I-RNA with poliovirus RNAresulted in over 90% inhibition of capsid protein synthesis. Activity ofI-9 RNA was approximately 50% of that observed with 1-7 or I-RNA. Higherconcentrations of I-9 RNA, however, inhibited viral protein synthesis tothe extent seen with I-7 RNA. As expected, I-8 RNA and I-4 RNA wereunable to inhibit translation of viral proteins. It should be noted thatsimilar amounts of intracellular poliovirus RNAs were detected in bothcells transfected with poliovirus RNA alone and those transfected with amixture of PV RNA and I-7 or I-9 RNAs, suggesting that the stability ofPV RNA is not altered significantly in cells containing I-RNA or itsderivatives.

EXAMPLE 11 Identification of Cellular Proteins that Interact with FullLength and Deleted I-RNAs

The 52 kDa I-RNA binding protein described above was shown to beidentical to the human La autoantigen and various other cellular proteinfactors were shown to bind to full-length or deleted I-RNAs.

For mobility shift experiments, fifty micrograms of HeLa S10 extract or10 pg of HeLa ribosomal salt wash (RSW) was preincubated at 30° C. for10 min with 4 pg of poly (dI-dC) (Pharmacia) in a 15 μl reaction mixturecontaining 5 mM HEPES (pH 7.6), 25 mM KCl, 2 mM MgCl2, 2 mM-DTT, 0.1 mMEDTA, 1.5 mM ATP, 2 mM GTP, and 3.8% glycerol. For competitionexperiments 100 fold molar excesses of unlabelled competitor RNAs wereadded to the reaction and incubated for 10 min at 30° C. Finally, 5 to10 fmol of labeled RNA probe was added to respective reaction mixturesand the incubation continued for another 20 min at 30° C. Threemicroliters of gel loading dye was added to the reaction mixture to afinal concentration of 5% glycerol and 0.02% each of bromophenol blueand xylene cyanol. For supershift assay the S10 extract was preincubatedwith either 2.5 μl of either nonimmune human sera or 2.5 μl of immunehuman sera against La protein on ice for 10 min, the respective32P-labeled RNA probe was then added to the reaction mixture andincubation was continued for another 20 min on ice. The RNA proteincomplexes were then analyzed on a 4% polyacrylamide gel (39:1 ratio ofacrylamide:bis) containing 5% glycerol in 0.5×TBE.

For UV-induced crosslinking and immunoprecipitation analyses,32P-labeled RNA-protein complexes generated as described above wereirradiated with a UV lamp (multi band UV) 254/366 nm (model UGL; 25 UVPInc.) at a distance of 2 to 3 cm for 15 min in a microtiter plate.Unbound RNAs were then digested with a mixture of 20 μg RNase A and 20units of RNase T1 at 37° C. for 15 min. For immunoprecipitation oflabeled complexes, 2-5 Al of either nonimmune human sera or immune humansera from a patient with lupus disease (standard reference La antibody)were added and kept on ice for 2h in the presence of 200 μ1×RIPA buffer[5 mM Tris(pH 7.9), 150 mM NaCl, 1% Triton-X 100, 0.1% SDS and 1% sodiumdeoxycholate). Five mg of protein A sepharose was then added to therespective reaction tubes, rocked in the cold room for 1h, thencentrifuged at 12,000 rpm for 5 min at 4° C. Beads were washed with1×RIPA three times to reduce nonspecific binding. Finally, resuspendedbeads in 1×SDS gel loading dye (50 mM Tris [pH 6.8], 100 mM DTT 2% SDS,0.1% BPB, 10% glycerol) were heated at 100° C. for 5 min. and analyzedon a SDS 14% polyacrylamide gel.

As shown in FIG. 12, a gel-retarded complex (denoted C) containinglabeled I-RNA and HeLa cell proteins was supershifted (denoted SC) by anantibody to the human La protein (FIG. 12, left gel, lanes 3 and 4). Asimilar complex formed with labeled I-RNA and purified recombinant Laprotein (lane 5, complex C) can also be supershifted with anti-Laantibody to the same relative position as found with HeLa cell extract.A second slower migrating complex was also observed with purified Laprotein (lane 5), the majority of which could not be supershifted withanti-La antibody (lane 6). These results suggest that complex C formedby incubating labeled I-RNA and HeLa cell extract contains Laautoantigen. To confirm that complex C indeed contains La protein,UV-crosslinking studies were performed with ³² P-labeled I-RNA or 5'-UTR(559-624 nt) probes using HeLa cell extract or purified La protein.UV-crosslinked complexes were then immunoprecipitated with anti-La ornonimmune sera and analyzed by SDS-PAGE. A 52 kDa UV-crosslinked proteinwas specifically immunoprecipitated by anti-La antibody when complexeswere formed with HeLa cell extract using either labeled I-RNA or UTRprobes (FIG. 12, right gel, lanes 2 and 5). This 52 kDa band co-migratedwith UV-crosslinked, anti-La immunoprecipitated complex formed byincubating purified La protein with ³² p I-RNA (lane 3) or ³² p 5'-UTR(lane 6). A prominent ˜120 kDa complex seen in lanes 2 and 5 was notspecific to La antibody as it could also be detected in lanes containingnonimmune serum. These results demonstrate that I-RNA interacts with thehuman La autoantigen.

The identity of the p52 protein bound by I-RNA was further confirmed bydemonstrating that inhibition of IRES-mediated translation by I-RNA isreversed by addition of La antigen. Poliovirus is known to inhibitcap-dependent translation of host cell mRNAs by proteolytically cleavingthe p220 component of the cap-binding protein complex. Therefore,extracts derived from virus-infected cells are only active incap-independent IRES-mediated translation but not cap-dependenttranslation.

To determine whether I-RNA-induced inhibition of IRES-mediatedtranslation can be specifically rescued by addition of exogenouspurified La protein, translation of p2CAT RNA (5-UTR-CAT) was performedin virus-infected cell extracts. Translation of p2 CAT RNA inPV-infected HeLa cell extract was inhibited significantly by I-RNA.Significant stimulation of viral 5'-UTR-mediated translation wasobserved when purified La protein was added to the infected cellextract. This is probably due to a limiting amount of La protein invirus-infected cells. Inhibition of translation mediated by I-RNA can bereversed by addition of purified La protein, almost to the extent seenin extract containing La protein alone. In contrast, addition of anequivalent amount of BSA failed to restore IRES-mediated translation. Asimilar result was observed when mock-infected extracts were usedinstead of virus-infected cell extracts.

Binding of various protein factors to 1-RNA mutants also was examined.Results presented above demonstrated differential activity of variousI-RNA mutants in inhibiting IRES-mediated translation. While I-7 and I-9RNAs were capable of inhibiting poliovirus IRES-mediated translation,I-4 and I-8 RNAs were almost totally inactive as translation inhibitors.To determine whether similar or different proteins were bound by theseRNAs, various labeled RNA probes were incubated with HeLa proteins andprotein-RNA complexes were examined by UV-crosslinking followingribonuclease digestion.

Two sources of HeLa proteins were used for these experiments, S10 andribosomal salt wash (RSW). HeLa Cell free extract (S10) and RSWpreparation. HeLa S10 cell extracts were prepared as describedhereinabove. Ribosomal salt wash from HeLa cells was prepared asdescribed by Brown and Ehrenfeld (33) with some modifications. Culturesof HeLa cells (4×10⁵ cells/ml) were harvested by centrifugation, washedthree times with cold isotonic buffer (35 mM HEPES, pH 7.5, 146 mM NaCl,11 mM glucose) and resuspended in two times packed cell volume of lysisbuffer (10 mM KC1 1.5 mM Mg acetate, 20 mM HEPES, pH 7.4, and 1 mM DTT)followed by incubation on ice for 10 min for swelling. Cells weredisrupted at O/C with 50 strokes in a type B dounce homogenizer. Afterdisruption extracts were centrifuged for 15 min at 10,000 rpm at 4/C inSorvall SS34 rotor to remove nuclei and mitochondrial fractions. Thesupernatant (S10 extract) was centrifuged at 50,000 rpm for 2h at 4/C ina Beckman Ti6O rotor. The ribosome pellet was resuspended at aconcentration of approximately 250 A260/ml in lysis buffer with gentleshaking on an ice bath. KCl concentration was then adjusted to 500 mMand the solution was stirred for 30 min on an ice bath. The resultingsolution was centrifuged for 2 h at 50,000 rpm at 4° C. The supernatant(salt wash) was then subjected to 0-70% ammonium sulfate precipitation.The pellet containing initiation factors was dissolved in low volume ofdialysis buffer (without glycerol) followed by overnight dialysis at 4°C. against the dialysis buffer containing 5 mM Tris(pH 7.5), 100 mM KCl,0.05 mM EDTA, 1 mM DTT and 5% glycerol. The dialysate was thencentrifuged at 10, 000 rpm for 10 min at 4° C. and the supernatant wasaliquoted in small volumes into several prechilled tubes and stored at-70° C.

When full-length labeled I-RNA was incubated with HeLa S10 extract, twomajor bands having approximate molecular weights of 52 kDa and 110 kDawere detected in addition to minor bands at 100, 70, 48 and 46 kDa. Whenribosomal salt wash proteins were used, the profile ofprotein-nucleotidyl complexes was significantly different from that withS10. First, the 110 kDa band was present in very low amounts inreactions containing RSW. Secondly, the 52 kDa protein was present as adoublet of 54-52 kDa and in relatively lower amounts compared to that inS10. Thirdly, new bands at approximately 80 and 37 kDa were apparent inRSW-containing reactions. In contrast to full-length I-RNA, when labeledtruncated I-RNAs were used in UV-crosslinking experiments, theprotein-nucleotidyl profile for each RNA was remarkably similar betweenS10 and RSW. While I-4 and I-8 RNAs bound mainly 70, 52, 48, 46 and 37kDa polypeptides, I-7 and I-9 RNAs interacted with a new band at 80 kDa.The 80 kDa band was more pronounced with I-7 RNA. Additionally, the 70kDa polypeptide bound by I-4 and I-8 RNAs was not detected with I-7 andI-9 RNAs. Another very high molecular weight polypeptide (running fasterthan the 220 kDa marker) was present only in reactions containing I-4and I-8 RNAs. Similarly, in experiments utilizing RSW the 100 kDapolypeptide was preferentially bound by I-7 and I-9 RNAs. In competitionexperiments, it was observed that both unlabeled I-7 and I-4 RNAssuccessfully competed with all major protein bands bound by labeled I-7and I-4 RNA probes. For example, all three polypeptides, 80 kDa, 52 kDaand 37 kDa, complexed to I-7 RNA were competed out with unlabeled I-7RNA and I-4 RNA but not with a non-specific RNA. Similarly with labeledI-4 RNA, the 70, 52 and 37 kDa bands were competed with unlabeled I-7and I-4 RNAs but not with a non-specific competitor. A higher molecularweight polypeptide was non-specifically bound to I-4 RNA as it could betotally competed out with a non-specific competitor. These resultsdemonstrate that while the active I-7 and inactive I-4 RNAs bind thesame two polypeptides (52 and 37 kDa), these two truncated I-RNAs differfrom each other in binding at least one polypeptide: I-7 RNA binds the80 kDa polypeptide, but I-4 RNA binds a 70 kDa polypeptide.

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By application of the principles described and exemplified above, it isapparent that one of ordinary skill in the art could design an effectiveinhibitor RNA or structural mimic thereof and determine optimumconditions of translation inhibition according to the present invention,for any desired target RNA on which translation is initiated at aninternal ribosome binding site by binding of one or more proteinfactors.

The entire disclosure of each publication cited herein is herebyincorporated herein by reference.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - <160> NUMBER OF SEQ ID NOS: 2                                               - <210> SEQ ID NO 1                                                           <211> LENGTH: 14                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Saccharomyces cerevisiae                                      - <400> SEQUENCE: 1                                                           #     14                                                                      - <210> SEQ ID NO 2                                                           <211> LENGTH: 60                                                              <212> TYPE: RNA                                                               <213> ORGANISM: Saccharomyces cerevisiae                                      - <400> SEQUENCE: 2                                                           - acggacgcgc ggguuucgaa guagcagaac agcgcaggaa cccggggaau gg - #aagcccgg         60                                                                          __________________________________________________________________________

We claim:
 1. A molecule that inhibits translation of an mRNA, whichtranslation is initiated at an internal ribosome entry site of said mRNAand requires binding of a protein factor to said site, said moleculeselectively binding to said factor, thereby preventing said factor frombinding to said site of said mRNA, wherein said molecule is an RNAoligonucleotide consisting of less than 35 nucleotides, and wherein thenucleotide sequence of said RNA oligonucleotide is5'GCGCGGGCAGCGCA3'(SEQ ID NO:1).
 2. An isolated nucleic acid moleculeselected from the group consisting of:a nucleic acid molecule with thenucleotide sequence GCGCGGGCAGCGA (SEQ. ID NO1); a nucleic acid moleculewith the nucleotide sequence depicted in SEQ. ID NO:2; and a nucleicacid molecule comprising 15 or more contiguous nucleotides of SEQ. IDNO:2.
 3. The nucleic acid molecule of claim 2, wherein said nucleic acidmolecule is an RNA molecule.
 4. An expression construct comprising DNAencoding the RNA molecule of claim 3 operably linked to an expressioncontrol sequence.